Pharmaceutically active agent complexes, polymer complexes, and compositions and methods involving the same

ABSTRACT

The present disclosure generally provides complexes including a pharmaceutically active agent and a functionalized polymer, wherein the functionalized polymer includes repeat units, the repeat units including ionizable repeat units having at least one ionizable side group and/or ionizable end group, a plurality of the at least one ionizable groups forming non-covalent bonds with the pharmaceutically active agent. Polymers which may be used to form such complexes as well as methods of making and using the complexes and related compositions are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and expressly incorporates byreference herein the entire disclosure of U.S. Provisional PatentApplication No. 61/913,827, filed Dec. 9, 2013.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to complexes including a pharmaceuticallyactive agent and a polymer, to compositions comprising such complexes,and to methods of making and using the same.

Background

The delivery of drugs, including injectable drug formulations, is oftenaccomplished in the art via the use of drug delivery depots. Followinginjection, drug delivery depots generally release drug in a slow andcontrolled manner. Drug delivery depots can also reduce and/or eliminatethe need for multiple injections. Such depots often include at leastpolymer, solvent, and drug. In some cases, the drug is highly soluble inthe depot and may leave the depot too quickly. In other cases, the drugis unstable in the depot.

The present disclosure addresses these and related issues and providesimproved compositions for the controlled release of pharmaceuticallyactive agents along with methods of making and using the same.

SUMMARY OF THE INVENTION

The present disclosure generally provides complexes including apharmaceutically active agent and a functionalized polymer, wherein thefunctionalized polymer includes repeat units, the repeat units includingionizable repeat units having at least one ionizable side group and/orionizable end group, a plurality of the at least one ionizable groupsforming non-covalent bonds with the pharmaceutically active agent.Polymers which may be used to form such complexes as well as methods ofmaking and using the complexes and related compositions are alsoprovided. Other features and advantages of the present disclosure willbe set forth in the description of invention that follows, and in partwill be apparent from the description or may be learned by practice ofthe invention. The invention will be realized and attained by thecompositions and methods particularly pointed out in the writtendescription and claims hereof.

Certain non-limiting aspects of the disclosure are provided below:

A first embodiment of the present disclosure is directed to a complexcomprising:

-   -   a pharmaceutically active agent, and    -   a functionalized polymer, the functionalized polymer comprising        repeat units, the repeat units comprising ionizable repeat units        comprising at least one ionizable side group, a plurality of the        at least one ionizable side groups forming a plurality of        non-covalent bonds with the pharmaceutically active agent,    -   wherein at least 10% of the repeat units comprise at least one        ionizable side group    -   wherein the functionalized polymer is optionally synthetic,    -   wherein the functionalized polymer is optionally a polyester,    -   wherein the functionalized polymer is optionally linear, and    -   wherein the functionalized polymer optionally has a weight        average molecular weight greater than 15,000 Daltons, as        measured by gel permeation chromatography.

A second embodiment of the present disclosure is directed to a complexcomprising:

-   -   a pharmaceutically active agent; and    -   a functionalized polymer, the functionalized polymer comprising        repeat units, the functionalized polymer comprising at least one        of: (a) ionizable repeat units comprising at least one ionizable        side group, wherein the at least one ionizable side group        comprises at least one member selected from ammonium,        carboxylate, hydrazinium, guanidinium, sulfate, sulfonate, and        phosphate; and (b) at least one ionizable end group;    -   wherein a plurality of the at least one ionizable groups form a        plurality of non-covalent bonds with the pharmaceutically active        agent,    -   wherein the functionalized polymer is optionally synthetic,    -   wherein the functionalized polymer is optionally a polyester,    -   wherein the functionalized polymer is optionally linear, and    -   wherein the functionalized polymer optionally has a weight        average molecular weight greater than 15,000 Daltons, as        measured by gel permeation chromatography.

A third embodiment of the present disclosure is directed to acomposition comprising:

-   -   a complex comprising:    -   a pharmaceutically active agent, and    -   a functionalized polymer complexed with the pharmaceutically        active agent through non-covalent bonding; and    -   a vehicle,    -   wherein the functionalized polymer is optionally synthetic,    -   wherein the functionalized polymer is optionally a polyester,    -   wherein the functionalized polymer is optionally linear, and    -   wherein the functionalized polymer optionally has a weight        average molecular weight greater than 15,000 Daltons, as        measured by gel permeation chromatography.

A fourth embodiment of the present disclosure is directed to a methodcomprising:

-   -   providing a precursor polymer comprising repeat units, the        repeat units comprising functionalizable repeat units comprising        at least one functionalizable side group;    -   obtaining a functionalized polymer by transforming, using click        chemistry, said functionalizable repeat units into ionizable        repeat units comprising at least one ionizable side group; and    -   combining the functionalized polymer with a pharmaceutically        active agent to form a complex in which a plurality of the at        least one ionizable side groups form a plurality of non-covalent        bonds with the pharmaceutically active agent.

A fifth embodiment of the present disclosure is directed to a methodcomprising:

-   -   combining a functionalized polymer with a pharmaceutically        active agent, the functionalized polymer comprising ionizable        repeat units comprising at least one ionizable side group, to        form a complex in which a plurality of the at least one        ionizable side groups form a plurality of non-covalent bonds        with the pharmaceutically active agent.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is further described in the description ofinvention that follows, in reference to the noted plurality ofnon-limiting drawings, wherein:

FIG. 1 is a graph showing the effect of polymer/peptide ratio on thecomplexation efficiency of exenatide and a GLP-1 analog.

FIG. 2 is a graph showing the effect of polymer/peptide ratio on thecomplexation efficiency of liraglutide with two differentamine-functionalized 50:50 copolymers.

FIG. 3 is a graph showing the dissolution of exenatide from anexenatide:amine-functionalized 50:50 copolymer complex relative to anexenatide:Zn/Protamine complex.

FIG. 4 is a graph showing the dissolution of a GLP-1 analog from aGLP-1:amine-functionalized 50:50 copolymer complex and aGLP-1:amine-functionalized homopolymer complex.

FIG. 5 is a graph showing the dissolution of risperidone from twodifferent mixtures of risperidone and carboxylate-functionalized 50:50copolymer in which the polymers have a molecular weight of 5.6 kDa(M_(w) of precursor polymer) (2A) and 12 kDa (M_(w) of precursorpolymer) (2B), respectively.

FIG. 6 is a graph showing the dissolution of liraglutide (AA) fromseveral liraglutide:amine-functionalized 50:50 copolymer complexes.Dissolution of liraglutide from a liraglutide:Zn/Protamine complex isshown for comparison.

FIG. 7 is a graph showing the dissolution of decitabine and azacytidinefrom their respective complexes with amine-functionalized 50:50copolymer.

FIG. 8 is a graph showing the dissolution of the GLP-1 analog from anon-click chemistry-based complex (A21 complex-PPT). Dissolution of theGLP-1 analog from a protamine complex and a supernatant obtainedfollowing precipitation of the complex (A21 complex-Supe) is shown forcomparison.

FIG. 9 is a graph showing the % cumulative release of decitabine for anattempted complexation with various amine or carboxylate functionalizedcopolymers.

FIG. 10 is a graph showing the % cumulative release of decitabine froman attempted complexation with a carboxylate functionalized copolymer.

FIG. 11 is a graph showing the dissolution of liraglutide per se (Lira)and liraglutide from two different liraglutide:amine-functionalized50:50 copolymer complexes (AFCP-1 and AFCP-2). The dissolution ofliraglutide from a liraglutide:Zn/protamine complex (Lira+Zn/Prot) isshown for comparison.

FIG. 12 is a graph showing the same data as FIG. 11 on a differentscale.

FIGS. 13 and 14 provide geometric mean data for the in vivo plasmaconcentration of liraglutide following administration of aqueous andBB:PLA vehicle suspended liraglutide complexes, respectively. Data foran aqueous solution of Liraglutide (Bachem) in 50 mM NH₄HCO₃, and aliraglutide:Zn/protamine complex in aqueous suspension or BB:PLA vehicleare provided for comparison as discussed in Example 16.

FIG. 15 is a graph showing the calculated BA of liraglutide fromtreatment Groups 1-6 of Example 16.

FIG. 16 is a graph showing the complexation efficiency of each ofAFCP-2, AFCP-3, AFCP-4, and AFCP-5 amine functionalized copolymers withrecombinant human growth hormone (rhGH).

FIG. 17 is a graph showing the complexation efficiency for AFCP-2 withrhGH at various weight ratios.

FIG. 18 is a graph showing the dissolution of rhGH from AFCP-2, AFCP-3and AFCP-5 complexes. Native rhGH is plotted as a control.

FIG. 19 is a graph showing the complexation efficiency of AFCP-2 aminefunctionalized copolymer with Liraglutide at various w/w ratios.

FIG. 20 is a graph showing the fraction precipitated for thecomplexation of Liraglutide with AFCP-2 and AFCP-3 based on the molarratio Liraglutide to AFCP-2 and AFCP-3.

FIG. 21 is a graph showing the dissolution of IgG from AFCP-2, AFCP-3,AFCP-4, and AFCP-5 complexes.

FIG. 22 is a graph showing the dissolution of somatostatin analogue froma CFCP-1 carboxylate functionalized copolymer complex.

FIG. 23 is a graph showing the dissolution of cromolyn from variousamine functionalized polymer complexes.

FIG. 24 provides reverse phase high pressure liquid chromatography(RPLC) spectra showing the stability of glatiramer bromide (uncomplexedpowder—top, complexed with AFCP-2—middle, and complexed with AFCP-2 inbenzyl benzoate (BB)—bottom) following gamma irradiation at a dose of 15kGy.

FIG. 25 provides RPLC spectra showing the stability of insulin(uncomplexed in BB—top and complexed with CFCP-5 in BB—bottom) followinggamma irradiation at a dose of 15 kGy.

FIG. 26 provides RPLC spectra showing the stability of liraglutide(uncomplexed powder—top, complexed with AFCP-2—middle, and complexedwith AFCP-2 in benzyl benzoate (BB)—bottom) following gamma irradiationat a dose of 15 kGy.

FIG. 27 provides RPLC spectra showing the stability of hGH (uncomplexedin BB—top and complexed with AFCP-2 in BB—bottom) following exposure togamma irradiation at a dose of 15 kGy.

FIG. 28 provides RPLC spectra showing the stability of uncomplexedglatiramer bromide (top) and uncomplexed insulin (bottom) in the absenceof gamma irradiation.

FIG. 29 provides RPLC spectra showing the stability of uncomplexedliraglutide (top) and uncomplexed human growth hormone (bottom) in theabsence of gamma irradiation.

DESCRIPTION OF THE INVENTION

Unless otherwise stated, a reference to a compound or component includesthe compound or component by itself, as well as in combination withother compounds or components, such as mixtures of compounds.

As used herein, the singular forms “a,” “an,” and “the” include theplural reference unless the context clearly dictates otherwise.

Before further discussion, a definition of the following terms will aidin the understanding of the present invention.

As used herein, the term “functionalized polymer” means a polymer thatcomprises at least one of: (a) ionizable repeat units comprising atleast one ionizable side group; and (b) at least one ionizable endgroup. Thus, a “functionalized polymer” comprises at least one ionizablegroup. In an embodiment, the “functionalized polymer” comprises at leastone ionizable repeat unit comprising at least one ionizable side group.

By “ionizable” or “ionizable group” is meant a moiety that is eitherionized or capable of ionization. For instance, an_ionizable group maybe ionized in an aqueous solution at a given pH but not at others.Accordingly, the term “ionizable group” encompasses a moiety which is inan ionized form, i.e., a charged group. By “ionizable” or “ionizablegroup” in a polymer described herein is meant a moiety of the polymerthat is either ionized or is capable of ionization to form an ionic bondwith the pharmaceutically active agent when the polymer is combined withthe pharmaceutically active agent to form a complex. As used herein, theterm “ionizable group” with reference to an “ionizable group” on apolymer encompasses both an “ionizable side group” on an ionizablerepeat unit comprised within the polymer and an “ionizable end group” onthe polymer. Examples of typical ionizable groups include ammonium,carboxylate, hydrazinium, guanidinium, sulfate, sulfonate, phosphonateand phosphate. Consistent with the above definition of “ionizablegroup”, each of the groups ammonium, carboxylate, hydrazinium,guanidinium, sulfate, sulfonate, phosphonate and phosphate includes itscorresponding uncharged (but ionizable) moiety, e.g. ammonium includesamino, carboxylate includes carboxylic acid, and so on. Ammoniumincludes primary, secondary and tertiary ammonium groups (i.e., groupsderived from primary, second and tertiary amines), as well as quaternaryammonium. Each such group may also be present one or more times in agiven ionizable group, such as in a given ionizable side chain. Forexample, each such group may be present once, twice or three or evenmore times. Thus, for example, dicarboxylate, tricarboxylate,diammonium, triammonium, polyamine, and polyamonium groups are allincluded. Ionizable groups of interest also include, e.g., succinate andspermine.

As used herein, the term “repeat unit” means a unit or residue in apolymer that is derived from a particular monomer. Typically the or eachrepeat unit is repeated multiple times in the chain of a particularpolymer molecule. A homopolymer comprises a plurality of identicalrepeat units. A copolymer comprises a plurality of different types ofrepeat unit, each of which is typically present multiple times in thechain of a particular polymer molecule.

As used herein, the term “precursor polymer” means a polymer that can beconverted into a “functionalized polymer” as defined herein bytransforming functionalizable repeat units in the precursor polymer intoionizable repeat units, for example using the methods describedelsewhere herein.

As used herein, the term “functionalizable side group” means a sidegroup that can be converted into an “ionizable side group” as definedherein, for example using the methods described elsewhere herein.

As used herein, the term “polymer intermediate” means a polymer that isan intermediate compound that is produced during the coversion of a“precursor polymer” as defined herein into a “functionalized polymer” asdefined herein.

As used herein, the term “hydrophilicity modifier” refers to a pendantgroup which is capable of changing the water solubility and/orhydrophilicity of a polymer to which the hydrophilicity modifier isbonded. Examples of pendant hydrophilicity modifiers includepolyethyleneglycols (PEG), hydroxyl groups and hydroxyalkyl groups.“Hydroxyalkyl” means an alkyl group, as defined herein, to which one ormore (such as one, two, three or four) hydroxy groups are attached.

As used herein, the term “alkyl” includes both saturated straight chainand branched alkyl groups. Preferably, an alkyl group is a C1-20 alkylgroup, more preferably a C1-15, more preferably still a C1-10 alkylgroup, more preferably still, a C1-5 alkyl group, and most preferably aC1-3 alkyl group such methyl. Particularly preferred alkyl groupsinclude, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl,tert-butyl, pentyl and hexyl. The term “alkylene” should be construedaccordingly.

As used herein, the term “alkenyl” refers to a group containing one ormore carbon-carbon double bonds, which may be branched or unbranched.Preferably the alkenyl group is a C2-20 alkenyl group, more preferably aC2-15 alkenyl group, more preferably still a C2-10 alkenyl group, orpreferably a C2-5 alkenyl group, and most preferably a C2-3 alkenylgroup. The term “alkenylene” should be construed accordingly.

As used herein, the term “alkynyl” refers to a carbon chain containingone or more triple bonds, which may be branched or unbranched.Preferably the alkynyl group is a C2-20 alkynyl group, more preferably aC2-15 alkynyl group, more preferably still a C2-10 alkynyl group, orpreferably a C2-5 alkynyl group and most preferably a C2-3 alkynylgroup. The term “alkynylene” should be construed accordingly.

As used herein, the term alkoxy refers to an alkyl group as definedherein that is attached to an oxygen atom.

As used herein, the term carbocyclyl includes a C3-7 carbocyclyl group,which is a non-aromatic saturated or unsaturated hydrocarbon ring havingfrom 3 to 7 carbon atoms. Preferably it is a saturated ormono-unsaturated hydrocarbon ring (i.e. a cycloalkyl moiety or acycloalkenyl moiety) having from 3 to 7 carbon atoms, more preferablyhaving from 5 to 6 carbon atoms. Examples include cyclopropyl,cyclobutyl, cyclopentyl and cyclohexyl and their mono-unsaturatedvariants. Particularly preferred carbocyclic groups are cyclopentyl andcyclohexyl. The term “carbocyclylene” should be construed accordingly.

As used herein, the term “aryl” includes a C6-10 aryl, which is amonocyclic or polycyclic 6- to 10-membered aromatic hydrocarbon ringsystem having from 6 to 10 carbon atoms. Phenyl is preferred. The term“arylene” should be construed accordingly.

As used herein, the term “heterocyclyl” includes a 5- to 10-memberedheterocyclyl group, which is a non-aromatic, saturated or unsaturated,monocyclic or polycyclic C5-10 carbocyclic ring system in which one ormore, for example 1, 2, 3 or 4, of the carbon atoms are replaced with amoiety selected from N, O, S, S(O) and S(O)₂. Preferably, the 5- to10-membered heterocyclyl group is a 5- to 6-membered ring. The term“heterocyclyene” should be construed accordingly.

Examples of heterocyclyl groups include azetidinyl, oxetanyl, thietanyl,pyrrolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl,thiazolidinyl, isothiazolidinyl, tetrahydrofuranyl, tetrahydrothienyl,tetrahydropyranyl, tetrahydrothiopyranyl, dithiolanyl, dioxolanyl,pyrazolidinyl, piperidinyl, piperazinyl, hexahydropyrimidinyl,methylenedioxyphenyl, ethylenedioxyphenyl, thiomorpholinyl,S-oxo-thiomorpholinyl, S,S-dioxo-thiomorpholinyl, morpholinyl,1,3-dioxolanyl, 1,4-dioxolanyl, trioxolanyl, trithianyl, imidazolinyl,pyranyl, pyrazolinyl, thioxolanyl, thioxothiazolidinyl,1H-pyrazol-5-(4H)-onyl, 1,3,4-thiadiazol-2(3H)-thionyl, oxopyrrolidinyl,oxothiazolidinyl, oxopyrazolidinyl, succinimido and maleimido groups andmoieties. Preferred heterocyclyl groups are pyrrolidinyl,imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl,isothiazolidinyl, tetrahydrofuranyl, tetrahydrothienyl,tetrahydropyranyl, tetrahydrothiopyranyl, dithiolanyl, dioxolanyl,pyrazolidinyl, piperidinyl, piperazinyl, hexahydropyrimidinyl,thiomorpholinyl and morpholinyl groups and moieties. More preferredheterocyclyl groups are tetrahydropyranyl, tetrahydrothiopyranyl,thiomorpholinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl,morpholinyl and pyrrolidinyl groups.

As used herein, the term “heteroaryl” includes a 5- to 10-memberedheteroaryl group, which is a monocyclic or polycyclic 5- to 10-memberedaromatic ring system, such as a 5- or 6-membered ring, containing atleast one heteroatom, for example 1, 2, 3 or 4 heteroatoms, selectedfrom 0, S and N. When the ring contains 4 heteroatoms these arepreferably all nitrogen atoms. The term “heteroarylene” should beconstrued accordingly.

Examples of monocyclic heteroaryl groups include thienyl, furyl,pyrrolyl, imidazolyl, thiazolyl, isothiazolyl, pyrazolyl, oxazolyl,isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, pyridinyl,pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and tetrazolyl groups.

Examples of polycyclic heteroaryl groups include benzothienyl,benzofuryl, benzimidazolyl, benzothiazolyl, benzisothiazolyl,benzoxazolyl, benzisoxazolyl, benztriazolyl, indolyl, isoindolyl andindazolyl groups. Preferred polycyclic groups include indolyl,isoindolyl, benzimidazolyl, indazolyl, benzofuryl, benzothienyl,benzoxazolyl, benzisoxazolyl, benzothiazolyl and benzisothiazolylgroups, more preferably benzimidazolyl, benzoxazolyl and benzothiazolyl,most preferably benzothiazolyl. However, monocyclic heteroaryl groupsare preferred.

Preferably the heteroaryl group is a 5- to 6-membered heteroaryl group.Particularly preferred heteroaryl groups are thienyl, pyrrolyl,imidazolyl, thiazolyl, isothiazolyl, pyrazolyl, oxazolyl, isoxazolyl,triazolyl, pyridinyl, pyridazinyl, pyrimidinyl and pyrazinyl groups.More preferred groups are thienyl, pyridinyl, pyridazinyl, pyrimidinyl,pyrazinyl, pyrrolyl and triazinyl, triazolyl, most preferably triazolyl.The term “triazolyl” is herein used interchangeably with “triazole” and,unless explicitly indicated to the contrary, refers to a 1,2,3-triazole.

As used herein, the term “optionally substituted” in the context ofoptionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted carbocyclyl, optionally substituted aryl, optionallysubstituted heterocyclyl, and optionally substituted heteroaryl means“unsubstituted or substituted”. “Substituted” means that one or morehydrogen atoms are replaced by substituents selected from halogen atomsand hydroxyl, —NH₂ and sulfonic acid groups. Typically from 1 to 10hydrogen atoms are replaced, more preferably 1 to 5, more preferablystill 1, 2 or 3 and most preferably 1 or 2, for example 1. Preferablyany given “substituted” group carries not more than 2 sulfonic acidsubstituents. Halogen atoms are preferred substituents. Preferably,though, the optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substituted alkoxy,optionally substituted carbocyclyl, optionally substituted aryl,optionally substituted heterocyclyl, and optionally substitutedheteroaryl are unsubstituted.

As used herein, the term “pharmaceutically active agent” means an agent,e.g., a protein, peptide, nucleic acid (including nucleotides,nucleosides and analogues thereof) or small molecule drug, that providesa desired pharmacological effect upon administration to a subject, e.g.,a human or a non-human animal, either alone or in combination with otheractive or inert components. Included in the above definition areprecursors, derivatives, analogues and prodrugs of pharmaceuticallyactive agents.

The terms “polypeptide” and “protein”, used interchangeably herein,refer to a polymeric form of amino acids of any length, which caninclude coded and non-coded amino acids, chemically or biochemicallymodified or derivatized amino acids, and polypeptides having modifiedpeptide backbones. The term includes fusion proteins, including, but notlimited to, fusion proteins with a heterologous amino acid sequence,fusions with heterologous and native leader sequences, with or withoutN-terminal methionine residues; immunologically tagged proteins; fusionproteins with detectable fusion partners, e.g., fusion proteinsincluding as a fusion partner a fluorescent protein, β-galactosidase,luciferase, etc.; and the like.

The terms “nucleic acid,” “nucleic acid molecule”, “oligonucleotide” and“polynucleotide” are used interchangeably and refer to a polymeric formof nucleotides of any length, either deoxyribonucleotides orribonucleotides, or compounds produced synthetically which can hybridizewith naturally occurring nucleic acids in a sequence specific mannersimilar to that of two naturally occurring nucleic acids, e.g., canparticipate in Watson-Crick base pairing interactions. Polynucleotidesmay have any three-dimensional structure, and may perform any function,known or unknown. Non-limiting examples of polynucleotides include agene, a gene fragment, exons, introns, messenger RNA (mRNA), transferRNA, ribosomal RNA, cDNA, recombinant polynucleotides, plasm ids,vectors, isolated DNA of any sequence, control regions, isolated RNA ofany sequence, nucleic acid probes, and primers.

As used herein “bioavailability” refers to the fraction of thepharmaceutically active agent dose that enters the systemic circulationfollowing administration.

As used herein, the terms “Glucagon-like-peptide-1” and “GLP-1” refer toa molecule having GLP-1 activity. One of ordinary skill in the art candetermine whether any given moiety has GLP-1 activity, as disclosed inU.S. Published Application No. 2010/0210505, which is incorporatedherein by reference. The term “GLP-1” includes native GLP-1 (GLP-1(7-37)OH or GLP-1 (7-36)NH2), GLP-1 analogs, GLP-1 derivatives, GLP-1biologically active fragments, extended GLP-1 (see, for example,International Patent Publication No. WO 03/058203, which is incorporatedherein by reference, in particular with respect to the extendedglucagon-like peptide-1 analogs described therein), exendin-4, exendin-4analogs, and exendin-4 derivatives comprising one or two cysteineresidues at particular positions as described in WO 2004/093823, whichis incorporated herein by reference.

When used to characterize a vehicle component or components as describedherein, the term “% w/w” refers to % by weight.

The term “click chemistry” comprises and identifies various groups ofchemical reactions characterized by particular properties such asrapidity, regioselectivity and high yield and having a highthermodynamic driving force. Among “click” reactions, cycloadditionreactions such as Diels-Alder reactions, and above all Huisgen1,3-dipolar cycloadditions, are particularly significant in the presentinvention. An example of a cycloaddition consists of a reaction in whichtwo unsaturated molecules react to form a cyclic compound with theformation of two new a bonds using π electrons.

As used herein, “cycloaddition” refers to a chemical reaction in whichtwo or more π (pi)-electron systems (e.g., unsaturated molecules orunsaturated parts of the same molecule) combine to form a cyclic productin which there is a net reduction of the bond multiplicity. In acycloaddition, the π (pi) electrons are used to form new sigma (a)bonds. The product of a cycloaddition is called an “adduct” or“cycloadduct”. Different types of cycloadditions are known in the artincluding, but not limited to, [3+2] cycloadditions and Diels-Alderreactions. [3+2] cycloadditions, which are also called 1,3-dipolarcycloadditions, occur between a 1,3-dipole and a dipolarophile and aretypically used for the construction of five-membered heterocyclic rings.The term “[3+2] cycloaddition” also encompasses “copperless” [3+2]cycloadditions between azides and cyclooctynes and difluorocyclooctynesdescribed by Bertozzi et al. J. Am. Chem. Soc., 2004, 126: 15046-15047.

A variety of methods are known in the art for determining the molecularweight of a polymer. Gel permeation chromatography may be utilized todetermine molecular weight as weight average molecular weight (Mw). Inaddition, number average molecular weight (Mn) may be calculated from ¹HNMR spectra. A suitable method may be selected at least in part based onthe approximate molecular weight of the polymer. For example,determination of molecular weight from NMR spectra may be suitable wherethe molecular weight of the polymer is less than about 45 kDa, while gelpermeation chromatography may be suitable for polymers having amolecular weight of greater than about 45 kDa.

An exemplary “gel permeation chromatography” (GPC) method utilizing anAgilent 1100 series liquid chromatography system is described below. Thesystem includes a pump, a solvent degasser, an automated injector, acolumn oven, and a differential refractive index detector. Agilent MixedD columns are used with polystyrene calibration standards.Tetrahydrofuran is used as the eluent. Both the columns and the detectorare maintained at 30° C. Calibration and calculation of polymermolecular weights are accomplished using the Chemstation® software.

The GPC method parameters are generally as follows:

Instrumentation: Agilent 1100 Series LC, equipped with a refractiveindex (RI) detector and solvent degasser.

Column Set: Agilent Mixed D® columns, 300×7.5 mm, Part No. PL1110-6504,two columns in series.

Eluent: 100% THF, stabilized, B&J Honeywell Cat. No. 341-4.

Calibrants: Agilent polystyrene EasiCal PS 2 standards, Part No.PL12010-0601, concentration 0.10% w/v

Sample Preparation: Weigh 0.045 to 0.055 grams of sample into a 20 mLvial and add 10 mL of THF (0.45-0.55% (w/v))

Instrument Conditions:

-   -   System Temperature: 30° C.    -   RI Detector: Polarity=positive    -   Flow rate: 1.0 mL/min    -   Injection Volume: 50 μL    -   Run Time: 25 min.

As discussed above, molecular weights (Mn) may be calculated using“NMR.” As an example, the spectra are obtained on a Bruker NMRspectrometer operating at 300 MHz. Mn is calculated from theintegrations of resonances assigned to polymer repeat units and polymerend groups. A sample calculation for a simplepoly(DL-lactide-co-glycolide) is shown below. Values of Mn for polymerand copolymers of α-chloro-ε-caprolactone can be calculated in a similarfashion.

The number of repeat units of DL-lactide (x), the number of repeat unitsof glycolide (y) and the value of the degree of polymerization (DP), andthe value of Mn from x and y are calculated from resonances assigned topolymer repeat units and polymer end groups. The structure below shows,the NMR assignments for the copolymer.

-   -   A=Intensity of 2 protons for Lactide Polymer (PLA) at 5.2 ppm    -   B=Intensity of 4 protons for Glycolide Polymer (PGY) at 4.95 ppm    -   C=Intensity of 2 (OCH2) protons for the 1-dodecanol end group        between 4.0 and 4.2 ppm    -   D=Intensity of 3 (CH3) protons for the 1-dodecanol end group        between 0.80 and 0.95 ppm

The values of x, y, DP, and Mn are calculated using the values of theintegrals from the NMR spectrum, A, B, and C:x=(A/2)/(C/2)=A/Cy=(B/4)/(C/2)=B/2CDP=x+yMn(Da)=144.13*x+116.07*y+186.34where 144.13 is the formula weight of DL-lactide in grams per mole,116.07 is the formula weight of glycolide in grams per mole, and 186.34is the formula weight of the 1-dodecanol residue in grams per mole.

Calculation of x, y, DP, and Mn using A, B, and D:x=(A/2)/(D/3)y=(B/4)/(D/3)DP=x+yMn(Da)=144.13*x+116.07*y+186.34where 144.13 is the formula weight of DL-lactide in grams per mole,116.07 is the formula weight of glycolide in grams per mole, and 186.34is the formula weight of the 1-dodecanol residue in grams per mole.

As used herein, the term “zero shear viscosity” means viscosity at zeroshear rate. A skilled artisan would be able to determine zero shearviscosity by measuring viscosity at low shear rate (e.g., around 1 sec⁻¹to 7 sec⁻¹) using a plate and cone viscometer (e.g., Brookfield ModelDV-III+(LV)) and then extrapolating a plot of viscosity versus shearrate to a shear rate of zero at a temperature of interest.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

To the extent the disclosure or the definition or usage of any termherein conflicts with the disclosure or the definition or usage of anyterm in an application or publication incorporated by reference herein,the instant application shall control.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

As an overview, the present disclosure relates to complexes including apharmaceutically active agent and a functionalized polymer, wherein thefunctionalized polymer includes repeat units, the repeat units includingionizable repeat units including at least one ionizable side group, aplurality of the at least one ionizable side groups forming a pluralityof non-covalent bonds with the pharmaceutically active agent. Among thepreferred embodiments of the present disclosure are embodiments in whichat least 10% of the repeat units include at least one ionizable sidegroup.

The present disclosure also provides complexes including apharmaceutically active agent and a functionalized polymer, thefunctionalized polymer including repeat units, the functionalizedpolymer including at least one of: (a) ionizable repeat units includingat least one ionizable side group, wherein the at least one ionizableside group includes at least one member selected from ammonium,carboxylate, hydrazinium, guanidinium, sulfate, sulfonate, andphosphate; and (b) at least one ionizable end group.

The present disclosure also provides compositions including a vehicleand complexes including a pharmaceutically active agent and afunctionalized polymer complexed with the pharmaceutically active agentthrough non-covalent bonding.

The present disclosure also provides methods including the steps of:providing a precursor polymer comprising repeat units, the repeat unitscomprising functionalizable repeat units comprising at least onefunctionalizable side group; obtaining a functionalized polymer bytransforming, using click chemistry, said functionalizable repeat unitsinto ionizable repeat units comprising at least one ionizable sidegroup; and combining the functionalized polymer with a pharmaceuticallyactive agent to form a complex in which a plurality of the at least oneionizable side groups form a plurality of non-covalent bonds with thepharmaceutically active agent.

The present disclosure also provides methods including the steps of:combining a functionalized polymer with a pharmaceutically active agent,the functionalized polymer comprising ionizable repeat units comprisingat least one ionizable side group, to form a complex in which aplurality of the at least one ionizable side groups form a plurality ofnon-covalent bonds with the pharmaceutically active agent.

Additional information related to the disclosure outlined above is nowprovided.

Complexes

The complexes of the present disclosure may be formed based onnon-covalent interaction (e.g., electrostatic, steric, hydrogen bonding,and van der Waals interactions) between a functionalized polymerincluding ionizable repeat units and/or ionizable end groups asdescribed herein and a pharmaceutically active agent. The complex may bea salt. Ideally, the complex reduces the solubility of thepharmaceutically active agent in the depot and/or in the body to prolongdrug release. The complex may also increase the stability of thepharmaceutically active agent, e.g., the radiation stability of thepharmaceutically active agent, either alone or when present in a depot.In addition to reducing the solubility, the complex achieves a vastlyincreased molecular weight such that its diffusion coefficient isreduced to slow movement of the pharmaceutically active agent. Thesolubility of the complex and its mobility in the environment can befurther modulated by the molecular weight of the complexing polymer andthe hydrophilic/hydrophobic nature of the polymer.

In some embodiments, the complex has a solubility of less than 0.01mg/mL in water at 25° C. at pH 7.4.

Functionalized Polymers for Complexes

To form the complexes of the present disclosure, functionalized polymershaving positively or negatively charged groups (or ionizable groupswhich may be ionized to positively or negatively charged groups) areprovided. Functionalized polymers having positively charged groups (orionizable groups which may be ionized to positively charged groups) aregenerally used to form complexes with negatively chargedpharmaceutically active agents and functionalized polymers havingnegatively charged groups (or ionizable groups which may be ionized tonegatively charged groups) are generally used to form complexes withpositively charged pharmaceutically active agents. Thus, in someembodiments the functionalized polymer has a net positive charge (or isionizable to provide a net positive charge). In other embodiments, thefunctionalized polymer has a net negative charge (or is ionizable toprovide a net negative charge). In some embodiments, the at least oneionizable side group is covalently bound to a precursor or intermediatepolymer through click chemistry to provide the functionalized polymer,for example the at least one ionizable side group is covalently bound tothe precursor or intermediate polymer through click chemistry catalyzedwith copper to provide the functionalized polymer.

In some embodiments, the ionizable repeat units of the functionalizedpolymer comprise one or more ionizable side groups that comprise anoptionally substituted heteroarylene ring, for example a 1,2,3-triazolering.

In some embodiments, the functionalized polymer functionalized withionizable groups is hydrophilic or water soluble. For instance, thefunctionalized polymer may have a water solubility ranging from 0.001mg/mL to 1000 mg/mL at 25° C. and pH 7.4, such as 0.01 mg/mL to 100mg/mL, 0.1 mg/mL to 10 mg/mL, or 1 mg/mL to 5 mg/m L. In someembodiments the functionalized polymer is biodegradable.

In some embodiments, the functionalized polymer is a polyester. Methodsfor producing polyesters are well known in the art. The functionalizedpolymers may include at least one repeat unit derived from a monomerselected from caprolactone, lactic acid, glycolic acid, lactide,glycolide, vinyl pyrrolidone, butyrolactone, and valerolactone, as wellas derivatives of these monomers that: (a) comprise ionizable sidegroups; and/or (b) comprise functionalizable side groups that can betransformed into ionizable side groups after polymerization has beeneffected (e.g. alpha-chlorocaprolactone). In some embodiments, thefunctionalized polymer is not derived from amino acid monomers. Thus, insome embodiments, the functionalized polymer is not a polyamino acid.

Examples of ionizable side groups include, but are not limited to,ammonium, carboxylate, hydrazinium, guanidinium, sulfate, sulfonate,phosphonate and phosphate.

The percentage of repeat units making up a functionalized polymer whichinclude at least one ionizable side group may vary. For example, thepercentage of repeat units making up a functionalized polymer and whichinclude at least one ionizable side group may be 100%, 50%, 25%, or12.5%. The percentage of repeat units making up a functionalized polymerand which include at least one ionizable side group may range from 10%to 90%, such as 20% to 80%, 30% to 70%, or 40% to 60%.

Accordingly, in some embodiments a complex according to the presentdisclosure, prepared using functionalized polymers such as thosedescribed herein, includes a functionalized polymer, the functionalizedpolymer including repeat units, the repeat units including ionizablerepeat units including at least one ionizable side group, a plurality ofthe at least one ionizable side groups forming a plurality ofnon-covalent bonds with the pharmaceutically active agent, wherein atleast 10% of the repeat units comprise at least one ionizable sidegroup, e.g., at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80% or at least 90%.

In some embodiments, less than 100% of the repeat units comprise atleast one ionizable side group. In some embodiments, the % of the repeatunits comprising at least one ionizable side group ranges from 10% to90%, e.g., 20% to 80%, 30% to 70%, or 40% to 60%. In some embodiments,the % of the repeat units comprising at least one ionizable side groupranges from 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%,60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100%.

The weight average molecular weight of the functionalized polymers isnot particularly limited and may range for example from 1000 Daltons to200,000 Daltons, as measured by gel permeation chromatography, e.g.,from 2000 Daltons to 50,000 Daltons, from 1000 Daltons to 100,000Daltons, from 1000 Daltons to 50,000 Daltons, from 1000 Daltons to40,000 Daltons, from 1000 Daltons to 30,000 Daltons, from 1000 Daltonsto 25,000 Daltons, from 1000 Daltons to 20,000 Daltons, from 1000Daltons to 10,000 Daltons, or from 1000 Daltons to 5000 Daltons.

In some embodiments, the functionalized polymer has a weight averagemolecular weight ranging from 100,000 Daltons to 200,000 Daltons, from50,000 Daltons to 200,000 Daltons, from 10,000 Daltons to 200,000Daltons, or from 5,000 Daltons to 200,000 Daltons, as measured by gelpermeation chromatography.

In some embodiments, the functionalized polymer has a number averagemolecular weight ranging from 5000 Daltons to 45,000 Daltons, asmeasured by NMR spectroscopy, e.g., from 10,000 Daltons to 45,000Daltons, from 20,000 Daltons to 45,000 Daltons, or from 30,000 Daltonsto 45,000 Daltons. In some embodiments, the functionalized polymer has anumber average molecular weight ranging from 10,000 Daltons to 20,000Daltons, or from 20,000 Daltons to 30,000 Daltons, as measured by NMRspectroscopy.

In some embodiments, the repeat units making up the functionalizedpolymer comprise repeat units comprising at least one pendanthydrophilicity modifier. Examples of these pendant hydrophilicitymodifiers include, but are not limited to, polyethyleneglycols (PEG),hydroxyl groups, and hydroxyalkyl groups.

In some embodiments, the functionalized polymer comprises at least oneionizable end group, for example the functionalized polymer may compriseionizable end groups at each of its ends. Examples of suitable ionizableend groups include ammonium, carboxylate, hydrazinium, guanidinium,sulfate, sulfonate, phosphonate and phosphate.

In some embodiments, the repeat units comprise repeat units of theformula (I):

wherein

m is an integer from 1 to 10, and

each R¹ and R² is independently selected from hydrogen, hydroxyl,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted carbocyclyl, optionally substituted aryl, optionallysubstituted heterocyclyl, optionally substituted heteroaryl, and aionizable side group.

The repeat units may, for example, comprise repeat units of the formula(I) wherein each R¹ and R² is independently selected from hydrogen, C₁₋₅alkyl and a ionizable side group. The repeat units may comprise repeatunits of the formula (I) wherein m is an integer from 1 to 5. The repeatunits may comprise repeat units of the formula (I) wherein one of the R¹and R²s is a ionizable side group and all of the remaining R¹ and R²sare not ionizable side groups. The repeat units may comprise repeatunits of the formula (I) wherein m is 5, one R¹ is a ionizable sidegroup and all of the remaining R¹ and R²s are hydrogen. Such repeatunits can readily be provided using the methods described herein byusing a caprolactone derivative as a monomer when preparing thefunctionalized polymer.

The ionizable side group in the formula (I) may comprise a positivelycharged side group or a negatively charged side group (or a side groupwhich ionizes to a positively charged or negatively charged side group).For example, the charged side group may comprise ammonium, carboxylate,hydrazinium, guanidinium, sulfate, sulfonate, phosphonate and phosphate.

The ionizable side group in the formula (I) may comprise an optionallysubstituted heteroarylene ring. For example, the ionizable side groupmay have the formula (II):

wherein R³ comprises a ionizable functional group. R³ may comprise atleast one selected from ammonium, carboxylate, guanidinium, sulfate,phosphonate and phosphate, either attached directly to the ring or via alinker group.

Further examples of repeat units of the formula (I), one or more ofwhich may be present in the functionalized polymer, include: repeatunits wherein m is 5 and all of the R¹ and R²s are hydrogen; repeatunits wherein m is 1, R¹ is hydrogen and R² is hydrogen; repeat unitswherein m is 1, R¹ is methyl and R² is hydrogen; repeat units wherein mis 2, the R¹ and R² alpha to the carbonyl group are each hydrogen, theR¹ beta to the carbonyl group is methyl and the R² beta to the carbonylgroup is hydrogen; repeat units wherein m is 3 and all of the R¹ and R²sare hydrogen; repeat units wherein m is 4, and all of the R¹ and R²s arehydrogen; and repeat units wherein m is 3, the R¹ and R² alpha to thecarbonyl group are each hydrogen, the R¹ and R² beta to the carbonylgroup are each hydrogen, and the R¹ gamma to the carbonyl group ismethyl and the R² gamma to the carbonyl group is hydrogen. Such repeatunits can readily be provided using the methods described herein byusing caprolactone, glycolic acid or glycolide, lactic acid or lactide,butyrolactone (e.g., beta-butyrolactone, gamma-butyrolactone, etc.) andvalerolactone (e.g., delta-valerolactone, gamma-valerolactone, etc.), orderivatives therefore, respectively as monomers when preparing thefunctionalized polymer. By “alpha” is meant the first position from adesignated carbon in an organic chemical structure at which an atom or aradical may be substituted. By “beta” is meant the second position froma designated carbon in an organic chemical structure at which an atom ora radical may be substituted. By “gamma” is meant the third positionfrom a designated carbon in an organic chemical structure at which anatom or a radical may be substituted. By “delta” is meant the fourthposition from a designated carbon in an organic chemical structure atwhich an atom or a radical may be substituted.

In some embodiments, the functionalized polymer is a homopolymer ofrepeat units of formula (I). Alternatively, the functionalized polymeris a copolymer comprising at least two different repeat units. Forexample, each of the at least two different repeat units in copolymermay be of formula (I) (but with different m, R¹ and/or R²s).

In some embodiments, the functionalized polymer includes one or morerepeat units. For example, the functionalized polymer may include 1 to10 repeat units, such as 1 to 7 repeat units, including 1 to 5 repeatunits, or 1 to 3 repeat units. In some instances, the functionalizedpolymer includes 1 to 3 repeat units, such as 1 repeat unit, 2 repeatunits or 3 repeat units. The repeat units may be any of the repeat unitsas described herein. Embodiments that include 1 repeat unit may also bereferred to as monomers rather than a polymer. In some instances, thefunctionalized polymer may include repeat units where each repeat unitis of the same formula, e.g., a homopolymer of the same repeat unit. Forexample, the functionalized polymer may include 2 repeat units whereeach repeat unit is of the same formula. In some instances, thefunctionalized polymer includes 3 repeat units where each repeat unit isof the same formula. In other embodiments, the functionalized polymer isa copolymer that includes at least two different repeat units. Forinstance, the functionalized polymer may include 2 repeat units, wherethe repeat units are different repeat units. In some cases, thefunctionalized polymer incldues 3 repeat units, where two repeat unitsare of the same formula and the third repeat unit is of a differentformula. In some instances, the functionalized polymer includes 3 repeatunits where each repeat unit is of a different formula. In embodimentsthat includes repeat units with different formulae, differentarrangements (i.e., permutations) of the repeat units are possible. Forexample, in embodiments that include 2 repeat units with differentformula (e.g., repeat unit 1 and repeat unit 2), the 2 repeat units maybe arranged in the polymer as: (repeat unit 1)-(repeat unit 2); or(repeat unit 2)-(repeat unit 1). Similarly, in embodiments that include3 repeat units where two repeat units are of the same formula and thethird repeat unit is of a different formula (e.g., repeat unit 1, repeatunit 1 and repeat unit 2; or repeat unit 1, repeat unit 2 and repeatunit 2), the 3 repeat units may be arranged in the polymer in one of 6different permutations:

(repeat unit 1)-(repeat unit 1)-(repeat unit 2);

-   -   (repeat unit 1)-(repeat unit 2)-(repeat unit 1);    -   (repeat unit 2)-(repeat unit 1)-(repeat unit 1);    -   (repeat unit 1)-(repeat unit 2)-(repeat unit 2);    -   (repeat unit 2)-(repeat unit 1)-(repeat unit 2); or    -   (repeat unit 2)-(repeat unit 2)-(repeat unit 1).

Similarly, in embodiments that include 3 repeat units where each repeatunit is of a different formula (e.g., repeat unit 1, repeat unit 2 andrepeat unit 3, the 3 repeat units may be arranged in the polymer in oneof 6 different permutations:

-   -   (repeat unit 1)-(repeat unit 2)-(repeat unit 3);    -   (repeat unit 1)-(repeat unit 3)-(repeat unit 2);    -   (repeat unit 2)-(repeat unit 1)-(repeat unit 3);    -   (repeat unit 2)-(repeat unit 3)-(repeat unit 1);    -   (repeat unit 3)-(repeat unit 1)-(repeat unit 2); or    -   (repeat unit 3)-(repeat unit 2)-(repeat unit 1).

The repeat units in the examples above (e.g., repeat unit 1, repeat unit2 and repeat unit 3) may be any of the repeat units as described herein.In certain embodiments, the functionalized polymer includes an optionalionizable group between the repeat units, such that a repeat unit islinked to an adjacent repeat unit through a linking ionizable unit. Inthese instances, the optional linking ionizable unit may be selectedfrom ammonium, carboxylate, hydrazinium, guanidinium, sulfate,sulfonate, phosphonate and phosphate.

An example of a functionalized polymer is poly(ε-caprolactone) (PCL)with amine pendant groups capable of complexing with a pharmaceuticallyactive agent (shown below in salt form):

Another example of a functionalized polymer is PCL copolymer with aminependant groups on a certain fraction of the repeat units capable ofcomplexing with a pharmaceutically active agent (shown below in saltform):

While the immediately preceding structure depicts a block copolymerstructure, it should be understood that, as with other block copolymerstructures described herein, the repeat units may alternatively berandomly distributed in the polymer.

Yet another example of a functionalized polymer is poly(ε-caprolactone)(PCL) with carboxylate pendant groups capable of complexing with apharmaceutically active agent (shown below in salt form):

A further example of a functionalized polymer is poly(ε-caprolactone)(PCL) with guanidinium pendant groups capable of complexing with apharmaceutically active agent (shown below in salt form):

Pharmaceutically Active Agents for Complexes

A wide variety of pharmaceutically active agents may be utilized in thecomplexes and compositions described herein, including, but not limitedto, peptides, proteins, and small molecules, e.g., small moleculeshaving a molecular weight less than 500 Daltons.

General classes of pharmaceutically active agents which may be utilizedinclude, for example, proteins, peptides, nucleic acids, nucleotides,nucleosides and analogues thereof, antigens, antibodies, and vaccines;as well as low molecular weight compounds.

Pharmaceutically active agents which may be utilized in the complexesand compositions disclosed herein include, but are not limited to,agents which act on the peripheral nerves, adrenergic receptors,cholinergic receptors, the skeletal muscles, the cardiovascular system,smooth muscles, the blood circulatory system, synaptic sites,neuroeffector junction sites, endocrine and hormone systems, theimmunological system, the reproductive system, the skeletal system,autacoid systems, the alimentary and excretory systems, the histaminesystem and the central nervous system.

Suitable pharmaceutically active agents may be selected, for example,from chemotherapeutic agents, epigenetic agents, proteasome inhibitors,adjuvant drugs, anti-emetics, appetite stimulants, anti-wasting agentsand high potency opioids.

Suitable pharmaceutically active agents may also be selected, forexample, from anti-neoplastic agents, cardiovascular agents, renalagents, gastrointestinal agents, rheumatologic agents and neurologicalagents among others.

Protein, Polypeptides and Peptides as Pharmaceutically Active Agents:

Proteins useful in the disclosed complexes and compositions may include,for example, molecules such as cytokines and their receptors, as well aschimeric proteins comprising cytokines or their receptors, including,for example tumor necrosis factor alpha and beta, their receptors andtheir derivatives; renin; growth hormones, including human growthhormone (e.g., rhGH), bovine growth hormone, methione-human growthhormone, des-phenylalanine human growth hormone, and porcine growthhormone; growth hormone releasing factor (GRF); octreotide, parathyroidand pituitary hormones; thyroid stimulating hormone; human pancreashormone releasing factor; lipoproteins; colchicine; prolactin;corticotrophin; thyrotropic hormone; oxytocin; vasopressin;somatostatin; lypressin; pancreozym in; leuprolide; alpha-1-antitrypsin;insulin; insulin analogs; insulin derivatives; insulin prodrugs;glargine; insulin A-chain; insulin B-chain; proinsulin; folliclestimulating hormone; calcitonin; luteinizing hormone; luteinizinghormone releasing hormone (LHRH); LHRH agonists and antagonists;glucagon; clotting factors such as factor VIIIC, factor IX, tissuefactor, and von Willebrands factor; anti-clotting factors such asProtein C; atrial natriuretic factor; lung surfactant; a plasminogenactivator other than a tissue-type plasminogen activator (t-PA), forexample a urokinase; bombesin; thrombin; hemopoietic growth factor;enkephalinase; RANTES (regulated on activation normally T-cell expressedand secreted); human macrophage inflammatory protein (MIP-1-alpha); aserum albumin such as human serum albumin; mullerian-inhibitingsubstance; relaxin A-chain; relaxin B-chain; prorelaxin; mousegonadotropin-associated peptide; chorionic gonadotropin; gonadotropinreleasing hormone; bovine somatotropin; porcine somatotropin; amicrobial protein, such as beta-lactamase; DNase; inhibin; activin;vascular endothelial growth factor (VEGF); receptors for hormones orgrowth factors; integrin; protein A or D; rheumatoid factors; aneurotrophic factor such as bone-derived neurotrophic factor (BDNF),neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nervegrowth factor such as NGF-β; platelet-derived growth factor (PDGF);fibroblast growth factor such as acidic FGF and basic FGF; epidermalgrowth factor (EGF); transforming growth factor (TGF) such as TGF-alphaand TGF-beta, including TGF-β-1, TGF-β-2, TGF-β-3, TGF-β-4, or TGF-β-5;insulin-like growth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I(brain IGF-I), insulin-like growth factor binding proteins; CD proteinssuch as CD-3, CD-4, CD-8, and CD-19; erythropoietin; osteoinductivefactors; immunotoxins; a bone morphogenetic protein (BMP); an interferonsuch as interferon-alpha (e.g., interferonα2A or interferonα2B), -beta,-gamma, -lambda and consensus interferon; colony stimulating factors(CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1to IL-10; superoxide dismutase; T-cell receptors; surface membraneproteins; decay accelerating factor; viral antigen such as, for example,a portion of the HIV-1 envelope glycoprotein, gp120, gp160 or fragmentsthereof; transport proteins; homing receptors; addressins; fertilityinhibitors such as the prostaglandins; fertility promoters; regulatoryproteins; antibodies and chimeric proteins, such as immunoadhesins;precursors, derivatives, prodrugs and analogues of these compounds, andpharmaceutically acceptable salts of these compounds, or theirprecursors, derivatives, prodrugs and analogues.

Suitable proteins or peptides may be native or recombinant and include,e.g., fusion proteins.

In some embodiments, the protein is a growth hormone, such as humangrowth hormone (hGH), recombinant human growth hormone (rhGH), bovinegrowth hormone, methione-human growth hormone, des-phenylalanine humangrowth hormone, and porcine growth hormone; insulin, insulin A-chain,insulin B-chain, and proinsulin; or a growth factor, such as vascularendothelial growth factor (VEGF), nerve growth factor (NGF),platelet-derived growth factor (PDGF), fibroblast growth factor (FGF),epidermal growth factor (EGF), transforming growth factor (TGF), andinsulin-like growth factor-I and -II (IGF-I and IGF-II).

Suitable peptides for use as the complexes and compositions disclosedherein include, but are not limited to, exenatide and Glucagon-likepeptide-1 (GLP-1) and precursors, derivatives, prodrugs and analoguesthereof. For instance, the peptide may be one or more of liraglutide,lixisenatide, albiglutide, dulaglutide, CJC-1134-PC, ACP-03, andsemaglutide.

Nucleic Acids as Pharmaceutically Active Agents: Nucleic acidpharmaceutically active agents include nucleic acids as well asprecursors, derivatives, prodrugs and analogues thereof, e.g.,therapeutic nucleotides, nucleosides and analogues thereof; therapeuticoligonucleotides; and therapeutic polynucleotides. Pharmaceuticallyactive agents selected from this group may find particular use asanticancer agents and antivirals. Suitable nucleic acid pharmaceuticallyactive agents may include for example ribozymes, antisenseoligodeoxynucleotides, aptamers and siRNA. Examples of suitablenucleoside analogues include, but are not limited to, cytarabine(araCTP), gemcitabine (dFdCTP), and floxuridine (FdUTP).

Other Pharmaceutically Active Agents: A variety of otherpharmaceutically active agents may be used in the compositions disclosedherein. Suitable compounds may include, but are not limited to,compounds directed to one or more of the following drug targets: Kringledomain, Carboxypeptidase, Carboxylic ester hydrolases, Glycosylases,Rhodopsin-like dopamine receptors, Rhodopsin-like adrenoceptors,Rhodopsin-like histamine receptors, Rhodopsin-like serotonin receptors,Rhodopsin-like short peptide receptors, Rhodopsin-like acetylcholinereceptors, Rhodopsin-like nucleotide-like receptors, Rhodopsin-likelipid-like ligand receptors, Rhodopsin-like melatonin receptors,Metalloprotease, Transporter ATPase, Carboxylic ester hydrolases,Peroxidase, Lipoxygenase, DOPA decarboxylase, A/G cyclase,Methyltransferases, Sulphonylurea receptors, other transporters (e.g.,Dopamine transporter, GABA transporter 1, Norepinephrine transporter,Potassium-transporting ATPase α-chain 1, Sodium-(potassium)-chloridecotransporter 2, Serotonin transporter, Synaptic vesicular aminetransporter, and Thiazide-sensitive sodium-chloride cotransporter),Electrochemical nucleoside transporter, Voltage-gated ion channels, GABAreceptors (Cys-Loop), Acetylcholine receptors (Cys-Loop), NMDAreceptors, 5-HT3 receptors (Cys-Loop), Ligand-gated ion channels Glu:kainite, AMPA Glu receptors, Acid-sensing ion channels aldosterone,Ryanodine receptors, Vitamin K epoxide reductase, MetGluR-like GABA_(B)receptors, Inwardly rectifying K⁺ channel, NPC1L1, MetGluR-likecalcium-sensing receptors, Aldehyde dehydrogenases, Tyrosine3-hydroxylase, Aldose reductase, Xanthine dehydrogenase, Ribonucleosidereductase, Dihydrofolate reductase, IMP dehydrogenase, Thioredoxinreductase, Dioxygenase, Inositol monophosphatase, Phosphodiesterases,Adenosine deaminase, Peptidylprolyl isomerases, Thymidylate synthase,Aminotransferases, Farnesyl diphosphate synthase, Protein kinases,Carbonic anhydrase, Tubulins, Troponin, Inhibitor of IκB kinase-β, Amineoxidases, Cyclooxygenases, Cytochrome P450s, Thyroxine 5-deiodinase,Steroid dehydrogenase, HMG-CoA reductase, Steroid reductases,Dihydroorotate oxidase, Epoxide hydrolase, Transporter ATPase,Translocator, Glycosyltransferases, Nuclear receptors NR3 receptors,Nuclear receptors: NR1 receptors, and Topoisomerase.

In some embodiments, pharmaceutically active agent is a compoundtargeting one of rhodopsin-like GPCRs, nuclear receptors, ligand-gatedion channels, voltage-gated ion channels, penicillin-binding protein,myeloperoxidase-like, sodium: neurotransmitter symporter family, type IIDNA topoisomerase, fibronectin type III, and cytochrome P450.

In some embodiments, the pharmaceutically active agent is an anticanceragent. Suitable anticancer agents include, but are not limited to,Actinomycin D, Alemtuzumab, Allopurinol sodium, Amifostine, Amsacrine,Anastrozole, Ara-CMP, Asparaginase, Azacytadine, Bendamustine,Bevacizumab, Bicalutimide, Bleomycin (e.g., Bleomycin A₂ and B₂),Bortezomib, Busulfan, Camptothecin sodium salt, Capecitabine,Carboplatin, Carmustine, Cetuximab, Chlorambucil, Cisplatin, Cladribine,Clofarabine, Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin,Daunorubicin, Daunorubicin liposomal, Dacarbazine, Decitabine,Docetaxel, Doxorubicin, Doxorubicin liposomal, Epirubicin, Estramustine,Etoposide, Etoposide phosphate, Exemestane, Floxuridine, Fludarabine,Fluadarabine phosphate, 5-Fluorouracil, Fotemustine, Fulvestrant,Gemcitabine, Goserelin, Hexamethylmelamine, Hydroxyurea, Idarubicin,Ifosfamide, Imatinib, Irinotecan, Ixabepilone, Lapatinib, Letrozole,Leuprolide acetate, Lomustine, Mechlorethamine, Melphalan,6-Mercaptopurine, Methotrexate, Mithramycin, Mitomycin C, Mitotane,Mitoxantrone, Nimustine, Ofatumumab, Oxaliplatin, Paclitaxel,Panitumumab, Pegaspargase, Pemetrexed, Pentostatin, Pertuzumab,Picoplatin, Pipobroman, Plerixafor, Procarbazine, Raltitrexed,Rituximab, Streptozocin, Temozolomide, Teniposide, 6-Thioguanine,Thiotepa, Topotecan, Trastuzumab, Treosulfan, Triethylenemelamine,Trimetrexate, Uracil Nitrogen Mustard, Valrubicin, Vinblastine,Vincristine, Vindesine, Vinorelbine, and analogues, precursors,derivatives and prodrugs thereof. It should be noted that two or more ofthe above compounds may be used in combination in the compositions ofthe present disclosure.

Pharmaceutically active agents of interest for use in the disclosedcompositions may also include opioids and derivatives thereof as well asopioid receptor agonists and antagonists, e.g., methadone, naltrexone,naloxone, nalbuphine, fentanyl, sufentanil, oxycodone, oxymorphone,hydrocodone, hydromorphone, and pharmaceutically acceptable salts andderivatives thereof.

In some embodiments the pharmaceutically active agent is a low molecularweight compound, e.g., a compound having a molecular weight of less thanor equal to about 800 Daltons, e.g., less than or equal to about 500Daltons. In some embodiments, the pharmaceutically active agent is acompound having a molecular weight ranging from 800 Daltons to 100Daltons, e.g., 700 Daltons to 200 Daltons, 600 Daltons to 300 Daltons,or 500 Daltons to 400 Daltons.

In some embodiments, the pharmaceutically active agent comprises atleast one member selected from a peptide, protein, and small molecule,the small molecule having a molecular weight less than 500 Daltons.

The pharmaceutically active agent may contain one functional group thatis capable of forming a non-covalent bond with the functionalizedpolymer. The pharmaceutically active agent may contain more than onefunctional group that is capable of forming non-covalent bonds with thefunctionalized polymer.

In some embodiments, the pharmaceutically active agent is stable inwater. For instance, when the pharmaceutically active agent is placed inwater at 25° C. for 1 hour, 12 hours, or 24 hours, the purity of thepharmaceutically active agent is degraded less than 5%, such as lessthan 3% or less than 2%.

The pharmaceutically active agent or pharmaceutically active agentcomplex may be present in any suitable concentration in the compositionsdisclosed herein. Suitable concentrations may vary depending on thepotency of the pharmaceutically active agent, pharmacokinetic half-life,etc. For example, the pharmaceutically active agent may be present in arange of from about 1% to about 50% by weight of the composition, e.g.,from about 5% to about 45%, from about 10% to about 40%, from about 15%to about 35%, or from about 20% to about 30% by weight of thecomposition. The complex including the pharmaceutically active agent maybe present at a concentration ranging from about 10 mg/mL to about 500mg/mL, such as from about 50 mg/mL to about 450 mg/mL, about 100 mg/mLto about 400 mg/mL, about 150 mg/mL to about 350 mg/mL, or about 200mg/mL to about 300 mg/m L.

In some embodiments, the complex comprising the functionalized polymerand pharmaceutically active agent has a solubility of less than 0.01mg/mL in water at 25° C. at pH 7.4.

In some embodiments, in the complex comprising the functionalizedpolymer and pharmaceutically active agent, the ratio of the amount ofthe pharmaceutically active agent to the amount of the functionalizedpolymer in the complex is from 1:1 to 1:10 by weight, e.g., from 1:1 to1:9 by weight, from 1:1 to 1:8 by weight, from 1:1 to 1:7 by weight,from 1:1 to 1:6 by weight, from 1:1 to 1:5 by weight, from 1:1 to 1:4 byweight, from 1:1 to 1:3 by weight, or from 1:1 to 1:2 by weight. In someembodiments, in the complex comprising the functionalized polymer andpharmaceutically active agent, the ratio of the amount of thepharmaceutically active agent to the amount of the functionalizedpolymer in the complex is from 1:1 to 1:2 by weight, from 1:2 to 1:3 byweight, from 1:3 to 1:4 by weight, from 1:4 to 1:5 by weight, from 1:5to 1:6 by weight, from 1:6 to 1:7 by weight, from 1:7 to 1:8 by weight,from 1:8 to 1:9 by weight, or from 1:9 to 1:10 by weight.

In one aspect, the complex may have a median particle size, as measuredby laser diffraction, of less than 10 micrometers, such as less than 5micrometers, less than 3 micrometers, less than 2 micrometers, or lessthan 1 micrometer. In some aspects, a process comprises milling thecomplex to achieve the desired particle size.

Pharmaceutical Compositions

The present disclosure provides pharmaceutical compositions including avehicle and a complex, the complex including a pharmaceutically activeagent and a functionalized polymer complexed with the pharmaceuticallyactive agent through non-covalent bonding.

Polymers

In some embodiments, the amount of the functionalized polymer present inthe composition is less than 50% by weight based on the total weight ofthe composition, e.g., less than 40%, less than 30%, less than 20%, lessthan 10%, or less than 5%.

In some embodiments, the amount of the functionalized polymer present inthe composition ranges from 5% by weight to 50% by weight based on thetotal weight of the composition, e.g., 10% by weight to 40% by weight,or 20% by weight to 30% by weight.

In some embodiments, the amount of the functionalized polymer present inthe composition ranges from 5% to 10% by weight based on the totalweight of the composition, e.g., 10% by weight to 20% by weight, 20% byweight to 30% by weight, 30% by weight to 40% by weight, or 40% byweight to 50% by weight.

Vehicle Polymers: In some embodiments, the vehicle includes a “vehicle”polymer, e.g., a biocompatible and/or biodegradable polymer. Asdescribed herein, the term “vehicle” polymer is used to distinguish thiscomponent from the functionalized polymer comprised in the complex. Thevehicle polymer is a polymer other than the functionalized polymercomprised in the complex. Suitable vehicle polymers may include, but arenot limited to, homopolymers, block-copolymers and random copolymers.Suitable polymers include those polymers or combinations of polymerswhich have solubility of at least about 20 weight %, 30 weight %, or 40weight % in a selected solvent or solvent combination. In someembodiments, suitable polymers include polymers having both hydrophilicand hydrophobic regions, e.g., an AB-type block copolymer composed ofhydrophobic and hydrophilic components. Such polymers may have atendency to form micelles when exposed to an aqueous environment as aresult of the amphiphilic character of the polymer. Suitable polymersmay include, but are not limited to, polylactides, polyglycolides,polycaprolactones, copolymers including any combination of two or moremonomers involved in the above, e.g., terpolymers of lactide, glycolideand ε-caprolactone, and mixtures including any combination of two ormore of the above. In other words, suitable polymers may also include,for example, polylactic acids, polyglycolic acids, polycaprolactones,copolymers including any combination of two or more monomers involved inthe above, e.g., terpolymers of lactic acid, glycolic acid andε-caprolactone, and mixtures including any combination of two or more ofthe above.

In some embodiments, a suitable vehicle polymer is polylactic acid(PLA), e.g., a PLA including an ionizable end-group (e.g., an acidend-group, e.g., in an acid-terminated PLA). Acid end-group PLAsinclude, e.g., lactate initiated PLAs described herein. In someembodiments, the PLA includes an unionizable end-group (e.g., an esterend-group, e.g., in an ester terminated PLA). Ester end-group PLAsinclude, but are not limited to, dodecanol-initiated (dd) PLAs describedherein. In some embodiments, the PLA is DL-PLA. In other embodiments,the biodegradable polymer is poly(lactic-co-glycolic acid) (PLGA), e.g.,dl-PLGA. In some embodiments, the PLGA includes an ionizable end-group,e.g., an acid end-group. Acid end-group PLGAs include, but are notlimited to, the glycolate initiated (ga) PLGAs described herein. In someembodiments, the PLGA includes an unionizable end-group, e.g., an esterend group. Ester end-group PLGAs include, but are not limited to,dodecanol initiated PLGAs described herein. In one embodiment, where thepolymer is a polycaprolactone, the polycaprolactone ispoly(c)caprolactone.

The vehicle polymer may be present in the vehicle in an amount rangingfrom about 5% to about 40% by weight of the vehicle, for example, fromabout 6% to about 35%, from about 7% to about 30%, from about 8% toabout 27%, from about 9% to about 26%, from about 10% to about 25%, fromabout 11% to about 24%, from about 12% to about 23%, from about 13% toabout 22%, from about 14% to about 21%, from about 15% to about 20%,from about 16% to about 19%, or at about 17% by weight of the vehicle.In some embodiments, the polymer is present in an amount of about 20% byweight of the vehicle.

In some embodiments, the vehicle polymer has a weight average molecularweight of from about 2 kDa to about 20 kDa, e.g., from about 2 kDa toabout 5 kDa, from about 2 kDa to about 10 kDa, or from about 2 kDa toabout 15 kDa. Additional embodiments include a biocompatible,biodegradable polymer having a weight average molecular weight of fromabout 5 kDa to about 15 kDa, e.g., about 10 kDa, e.g., as determined bygel permeation chromatography or NMR spectroscopy.

In some embodiments, the pharmaceutical composition, which includesvehicle polymer, has a low amount of functionalized polymer. Forinstance, the amount of functionalized polymer in the pharmaceuticalcomposition may be less than 50 wt %, such as less than 40 wt %, lessthan 30 wt %, less than 20 wt %, less than 10 wt % or less than 5 wt %,and may range from 5 wt % to 50 wt %, based on total weight of thepharmaceutical composition.

Also, the pharmaceutical composition may be free of polymer other thanthe functionalized polymer, e.g., free of vehicle polymer.

Solvents

Vehicle Solvents: In some embodiments the vehicle includes one or moresolvents in addition to, or to the exclusion of, one or more vehiclepolymers as discussed herein. In some embodiments, the solvent ispresent in the vehicle in an amount ranging from 60% to 100% by weightof the vehicle, e.g., from 70% to 100%, from 80% to 100% or from 90% to100%.

In some embodiments, the solvent includes at least one hydrophilic/polarsolvent. In addition to, or as an alternative to the at least onehydrophilic/polar solvent, the solvent may include at least onehydrophobic solvent.

In some embodiments, the solvent includes at least one member selectedfrom water, a buffered aqueous system, dimethylsulfoxide (DMSO), benzylalcohol (BA), benzyl benzoate (BB), hydrogenated castor oil,polyethoxylated castor oil, dimethylacetamide, ethanol, ethyl acetate,glycofurol, isopropyl myristate, ethyl benzoate, caprylic/caprictriglyceride, n-methyl-pyrrolidone (NMP), propylene glycolmonocaprylate, propylene carbonate, 2-pyrrolidone, triacetin, andtriethyl citrate.

In some embodiments, the solvent is a benign vehicle solvent, such as anaqueous medium, BA, BB, DMSO, ethanol, ethyl acetate, glycofurol,propylene carbonate or NMP. The complexes may therefore improveinjection site compatibility, which is sometimes an issue with drugdepots.

A wide variety of vehicle solvents may be used. Water or bufferedaqueous systems may suffice. Thus, the vehicle solvent may involveaqueous or non-aqueous systems comprising at least one of the following:dimethylsulfoxide (DMSO), benzyl alcohol (BA), benzyl benzoate (BB),Cremophor EL (polyethoxylated castor oil), dimethylacetamide, ethanol,ethyl acetate, glycofurol, isopropyl myristate, ethyl benzoate, Miglyol810 (caprylic/capric triglyceride), n-methyl-pyrrolidone, Capryol 90(propylene glycol monocaprylate), propylene carbonate, 2-pyrrolidone,triacetin, and triethyl citrate.

In some embodiments, the pharmaceutical composition is free of organicsolvent. In other embodiments the pharmaceutical composition has arelatively low amount of organic solvent. For instance, the amount oforganic solvent in the pharmaceutical composition may be less than 10 wt%, such as less than 5 wt % or less than 1 wt %, based on total weightof the pharmaceutical composition. The amount of organic solvent mayrange from 60 wt % to 95 wt %, based on total weight of thepharmaceutical composition, e.g., 65 wt % to 90 wt %, 70 wt % to 85 wt%, or 75 wt % to 80 wt %.

In some embodiments, the vehicle includes a hydrophobic solvent.Hydrophobic solvents suitable for use in the vehicles of the presentdisclosure may be selected based on their ability to solubilize apolymer component of the vehicles described herein. Hydrophobic solventscan be characterized as being insoluble or substantially insoluble inwater. For example, suitable hydrophobic solvents have solubility inwater of less than 5% by weight, less than 4% by weight, less than 3% byweight, less than 2% by weight or less than 1% by weight, e.g. asmeasured at 25° C. A suitable hydrophobic solvent may also becharacterized as one which has a solubility in water of about 5% orless, about 4% or less, about 3% or less, about 2% or less, or about 1%or less, at 25° C. For example, in some embodiments, a suitablehydrophobic solvent has a solubility in water of from about 1% to about7%, from about 1% to about 6%, from about 1% to about 5%, from about 1%to about 4%, from about 1% to about 3%, and from about 1% to about 2%,at 25° C. A suitable hydrophobic solvent may also be characterized as asolvent in which water has limited solubility, e.g., a solvent in whichwater has solubility of less than 10% by weight, less than 5% by weight,or less than 1% by weight, at 25° C.

In some embodiments, suitable hydrophobic solvents include derivativesof benzoic acid including, but not limited to, benzyl alcohol, methylbenzoate, ethyl benzoate, n-propyl benzoate, isopropyl benzoate, butylbenzoate, isobutyl benzoate, sec-butyl benzoate, tert-butyl benzoate,isoamyl benzoate and benzyl benzoate.

In some embodiments, benzyl benzoate is selected as the hydrophobicsolvent for use in the vehicles of the present disclosure.

A suitable solvent may be a single solvent selected from among thefollowing or a combination of two or more of the following: benzylalcohol, benzyl benzoate, ethyl benzoate, and ethanol.

Where the solvent is a hydrophobic solvent, it may be used incombination with one or more additional solvents, e.g., one or morehydrophobic solvents and/or one or more polar/hydrophilic solvents.

In some embodiments, the compositions include a single hydrophobicsolvent as described herein without including any additional solvents.

Where the solvent is a polar/hydrophilic solvent, it may be used alonein the vehicle or in combination with one or more hydrophobic solventsas described herein. In some embodiments, where the polar/hydrophilicsolvent is used in combination with one or more hydrophobic solvents asdescribed herein, the polar/hydrophilic solvent is present in arelatively small amount relative to the hydrophobic solvent, e.g., lessthan 5% (e.g., less than 4%, less than 3%, less than 2%, or less than1%) by weight of the vehicle. For example, a polar/hydrophilic solventmay be present in the vehicle in an amount of from about 5% to about 1%(e.g., from about 4% to about 1%, from about 3% to about 1%, or fromabout 2% to about 1%) by weight of the vehicle.

In some embodiments, the total amount of solvent in the composition isat least 70% by weight based on the total weight of the composition,e.g., at least 80% by weight, at least 90% by weight, at least 95% byweight, or at least 99% by weight. In some embodiments, the total amountof solvent in the composition ranges from 70% to 99% by weight based onthe total weight of the composition, e.g., 75% to 99%, 80% to 99%, 90%to 99%, or 95% to 99%. The amount of solvent typically ranges from 60 wt% to 95 wt %, based on total weight of the pharmaceutical composition.

In some embodiments, the polymer-pharmaceutically active agent complexmay allow for the use of reduced amounts of solvent in thepharmaceutical composition. For instance, the amount of solvent in thepharmaceutical composition may be less than 10 wt %, such as less than 5wt % or less than 1 wt %, based on total weight of the pharmaceuticalcomposition.

Since the functionalized polymer of the disclosed compositions isdirectly complexed with the pharmaceutically active agent, thecompositions may be free of other complexing agents, such as protamine,Zn²⁺, carboxymethylcellulose (CMC), or other stabilizers. In othercases, the composition comprises less than 5 wt %, less than 4 wt %,less than 3 wt %, less than 2 wt %, less than 1 wt %, less than 0.5 wt%, less than 0.2 wt %, or less than 0.1 wt % of other complexing agents,such as protamine, Zn²⁺, carboxymethylcellulose (CMC), or otherstabilizers.

Excipients

In some embodiments, the pharmaceutical compositions according to thepresent disclosure include one or more excipients, e.g., stabilizers,dyes, fillers, preservatives, buffering agents, antioxidants, wettingagents, anti-foaming agents, surfactants, and the like. Excipients mayinclude, e.g., sucrose, polysorbate, methionine, mannitol, trehalose,etc. An example of a preferred excipient is sucrose acetate isobutyrate(SAIB).

Methionine may be included in a composition of the present disclosure asan antioxidant, and in some embodiments sucrose is included as astabilizer. Methionine may be combined with a pharmaceutically activeagent complex as described herein to form a radiation stable powder or aradiation stable composition.

In some embodiments, the pharmaceutical composition has a low excipientto pharmaceutically active agent ratio. For instance, the weight ratioof excipient to pharmaceutically active agent may range from 1:10 to1:1000, e.g., 1:50 to 1:1000, 1:100 to 1:1000, or 1:500 to 1:1000.

Additional description of vehicles and vehicle components which may beused in connection with the disclosed compositions is provided in U.S.Patent Application Publication No. 2012/0225033, filed Nov. 23, 2011,which application is incorporated by reference herein in its entiretyand for all purposes.

Radiation-Sterilized Compositions

As discussed briefly above, methionine may be combined with apharmaceutically active agent complex as described herein to form aradiation stable powder or a radiation stable composition. Additionaldescription of radiation-stable compositions into which the complexes ofthe present disclosure may be incorporated is provided in InternationalPCT Application No. WO2013/078396, filed Nov. 21, 2012, the disclosureof which is incorporated by reference herein in its entirety and for allpurposes.

For example, a radiation stable composition may be prepared by 1)combining a biodegradable polymer and a hydrophobic solvent to form asingle-phase vehicle of the composition, wherein the biodegradablepolymer is included in an amount of from about 5% to about 40% by weightof the vehicle, and the hydrophobic solvent is included in an amount offrom about 95% to about 60% by weight of the vehicle; 2) dispersing acomplex according to the present disclosure in the vehicle to form thecomposition; and irradiating the composition with ionizing radiation,wherein the pharmaceutically active agent maintains a purity of about90% or greater when stored at 25° C. for a period of 24 hours afterirradiation. In some embodiments antioxidant is added to the compositionprior to irradiating the composition with ionizing radiation, e.g., at adose of 10 kGy to 25 kGy. In some embodiments, the antioxidant is addedin an amount ranging from about 1 wt % to about 45 wt %, relative to theamount of pharmaceutically active agent. In some embodiments, methionineis added to the composition prior to irradiating the composition, e.g.,in an amount of from 0.1 wt % to about 45 wt %, relative to the amountof pharmaceutically active agent.

Pharmaceutical Composition Properties

In one aspect, the complex is present in the pharmaceutical compositionin the form of particles. The particles optionally include at least oneexcipient in addition to the complex. The particles may have a medianparticle size, as measured by laser diffraction, of less than 10micrometers, such as less than 5 micrometers, less than 3 micrometers,less than 2 micrometers, or less than 1 micrometer. In some aspects, aprocess comprises milling the complex to achieve the desired particlesize.

The pharmaceutical compositions of the present invention typically haverelatively low viscosity. For instance, the pharmaceutical compositionsmay have a zero shear viscosity less than 1,200 centipoise (cP), e.g.,less than 1000 cP, less than 500 cP, less than 100 cP, less than 50 cP,or less than 10 cP, at 25° C. The zero shear viscosity of thepharmaceutical compositions typically range from about 10 cP to about1200 cP, e.g., about 50 cP to about 1000 cP, or about 100 cP to about500 cP, at 25° C.

Surprisingly, the disclosed pharmaceutical compositions typicallydemonstrate good syringeability and injectability while providing forsustained release of the pharmaceutically active agent in-vivo withminimal burst. Syringeability and injectability may be characterized bythe time it takes to inject a known volume of the pharmaceuticalcomposition through a syringe of known size fitted with a relativelysmall gauge needle, e.g., a 1-5 mL syringe fitted with a needle having agauge of about 21 to about 27. In some embodiments, the pharmaceuticalcompositions of the present disclosure may be characterized as havinggood syringeability and injectability based on their ability to beinjected through a 1 ml syringe fitted with an approximately 0.5 inneedle having a gauge of about 21 to about 27, wherein a 0.5 ml volumeof the pharmaceutical composition can be injected in less than 25 sec(e.g., less than 20 sec., less than 15 sec, less than 10 sec, or lessthan 5 sec) at 25° C. with the application of a 5 to 10 lb-force. Insome embodiments, under the above conditions, the pharmaceuticalcomposition can be injected in a range of from about 1.5 sec to about 25sec, e.g., from about 1.5 sec to about 20 sec, from about 1.5 sec toabout 15 sec, from about 1.5 sec to about 10 sec, or from about 1.5 secto about 5 sec.

In addition to good injectability and syringeability as describedherein, in some embodiments, the pharmaceutical compositions of thepresent disclosure demonstrate minimal burst and sustained delivery ofpharmaceutically active agent over time. “Minimal burst” may becharacterized in terms of Cmax/Cmin, wherein the acceptable Cmax/Cminupper limit may vary depending on the pharmaceutically active agent tobe delivered. In some embodiments, the weight % of pharmaceuticallyactive agent released as burst over the first 24 hours is less than 30%of the total amount released over one week, e.g., less than 20% or lessthan 10%, of the total amount released over one week. For example, theweight % of pharmaceutically active agent released as burst over thefirst 24 hours may be less than about 30%, less than about 20%, lessthan about 20%, or less than about 10%, of the total amount releasedover one week. In some embodiments, the weight % of pharmaceuticallyactive agent released as burst over the first 24 hours is less than 10%of the total amount released over one month, e.g., less than 8% or lessthan 5%, of the total amount released over one month. For example, theweight % of pharmaceutically active agent released as burst over thefirst 24 hours may be less than 10% to about 8% or from about 8% toabout 5%, of the total amount released over one month. In someembodiments, the Cmax to Cmin ratio of the pharmaceutically activeagent, as measured over 28 days, 21 days, 14 days, or 7 days afteradministration, may range from 2 to 40, such as from 5 to 30, or 10 to20. In some embodiments, the Cmax to Cmin ratio of the pharmaceuticallyactive agent, as measured over 7 days after administration, may be lessthan 10, less than 5, less than 4, or less than 2. As used herein,“sustained delivery” refers to durations which are at least severalfold, e.g., at least 5 fold to at least 10 fold, longer than theduration obtained from a single dose of an immediate-release (IR)formulation of the same pharmaceutically active agent (determined byAdsorption, Distribution, Metabolism, and Excretion (ADME)characteristics of the pharmaceutically active agent itself).

As mentioned above, the disclosed biodegradable compositions provide forsustained release of the pharmaceutically active agent in-vivo withminimal burst effect in addition to possessing good injectability,syringeability and chemical stability as discussed above. This is anunexpected and surprising result as currently available formulationsgenerally provide either controlled release orinjectability/syringeability but not both. For example, commerciallyavailable depot formulations may rely on the formation of an extremelyviscous polymer matrix to provide controlled release of apharmaceutically active agent. However, such formulations have poorinjectability/syringeability due to the viscous nature of the depot.Alternatively, other commercially available formulations utilizevehicles which may have good injectability/syringeability due to ahigh-solvent content but poor control over release of thepharmaceutically active agent. Moreover, one would expect a lowviscosity liquid composition such as those disclosed herein to have poorrelease kinetics in the form of a substantial burst effect and anexponentially declining delivery profile. Contrary to this expectation,the present compositions demonstrate low burst effect and good controlover release of the pharmaceutically active agent over a period of oneday to one month or longer.

Administration of Pharmaceutical Compositions

As discussed previously herein, the disclosed compositions typicallypossess low viscosity along with good injectability and syringeabilitymaking them well suited for delivery via a syringe (e.g., a 1-5 mLsyringe) with a needle, e.g., 18 gauge to 27 gauge, such as a narrowgauge needle, e.g., 21 to 27 gauge. In addition, the pharmaceuticalcompositions may also be delivered via one or more pen injectors orneedleless injectors known in the art.

The pharmaceutical formulations of the present invention allow for lowinjection volume. For instance, the injection volume may be less than 1mL, such as less than 750 μL, less than 500 μL, or less than 250 μL.

Suitable routes of administration include, but are not limited to,subcutaneous injection and intramuscular injection. Suitable routes ofadministration also include, for example, intra-articular andintra-ocular, e.g., intra-vitreal, administration for local delivery.

The pharmaceutical compositions disclosed herein may also find use inoral compositions, e.g., compositions delivered in a gel-cap (soft orhard) or as a mouthwash.

The compositions of the present disclosure may be formulated such that adesired pharmacological effect is achieved via administration on aperiodic basis. For example, the compositions may be formulated foradministration on a daily, weekly or monthly basis.

The actual dose of the pharmaceutically active agent to be administeredwill vary depending on the pharmaceutically active agent, the conditionbeing treated, as well as the age, weight, and general condition of thesubject as well as the severity of the condition being treated, and thejudgment of the health care professional. Therapeutically effectiveamounts are known to those skilled in the art and/or are described inthe pertinent reference texts and literature.

For example, in the case of proteins and peptides pharmaceuticallyactive agents, the pharmaceutically active agent will typically bedelivered such that plasma levels of the pharmaceutically active agentare within a range of about 1 picomole/liter to about 1 micromole/liter,such as about 5 picomoles/liter to about 1 nanomole/liter or about 50picomoles/liter to about 200 picomoles/liter. On a weight basis, atherapeutically effective dosage amount of protein or peptide willtypically range from about 0.01 mg per day to about 1000 mg per day foran adult. For example, peptide or protein dosages may range from about0.1 mg per day to about 100 mg per day, or from about 1.0 mg per day toabout 10 mg/day.

In some embodiments, a suitable low molecular weight compound may becharacterized as one which can provide the desired therapeutic effectwith a dose of less than or equal to about 30 mg/day as delivered from adepot administered once a week, or a dose of less than or equal to about10 mg/day as delivered from a depot administered once a month. Forexample, a suitable low molecular weight compound may be one which canprovide the desired therapeutic effect with a dose of less than about 30mg/day, e.g., less than about 25 mg/day, less than about 20 mg/day, lessthan about 15 mg/day, less than about 10 mg/day, less than about 5mg/day or less than about 1 mg/day as delivered from a depotadministered once a week. In some embodiments, a suitable low molecularweight compound is one which can provide the desired therapeutic effectwith a dose of from about 1 mg/day to about 30 mg/day, e.g., from about5 mg/day to about 25 mg/day, or from about 10 mg/day to about 20 mg/dayas delivered from a depot administered once a week. In some embodiments,the dose may range from 0.1 mg/kg to 10 mg/kg, such as 0.5 mg/kg to 5mg/kg or 1 mg/kg to 3 mg/kg.

Similarly, a suitable low molecular weight compound may be one which canprovide the desired therapeutic effect with a dose of less than about 10mg/day, less than about 9 mg/day, less than about 8 mg/day, less thanabout 7 mg/day, less than about 6 mg/day, less than about 5 mg/day, lessthan about 4 mg/day, less than about 3 mg/day, less than about 2 mg/dayor less than about 1 mg/day as delivered from a depot administered oncea month. In some embodiments, a suitable low molecular weight compoundmay be one which can provide the desired therapeutic effect with a doseof from about 1 mg/day to about 10 mg/day, e.g., from about 2 mg/day toabout 9 mg/day, from about 3 mg/day to about 8 mg/day, from about 4mg/day to about 7 mg/day, or from about 5 mg/day to about 6 mg/day asdelivered from a depot administered once a month.

In some embodiments, the Cmax to Cmin ratio of the pharmaceutical activeagent, as measured over 28 days, 21 days, 14 days, or 7 days afteradministration, typically ranges from 2 to 40, such as from 5 to 30 or10 to 20, and may be less than 10, less than 5, less than 4, or lessthan 2.

In some embodiments, e.g., where the composition may have been instorage for a period of time prior to injection, the composition may bemixed, e.g., via shaking, prior to administration to ensure that thecomplex comprising pharmaceutically active agent is sufficientlydispersed in the vehicle carrier.

In some embodiments the pharmaceutical compositions disclosed herein (orcomponents thereof) are sterilized prior to use, e.g., via theapplication of a sterilizing dose of ionizing radiation. For example, inone embodiment one or both of the complex and pharmaceutical compositionas disclosed herein are sterilized with ionizing radiation, e.g., gammaradiation, e-beam radiation, or x-ray radiation.

One of ordinary skill in the art will be able to determine anappropriate sterilizing dose of radiation based on a variety of factorsincluding, e.g., the type of radiation, the shape, size, and/orcomposition of the material to be sterilized, the desired level ofsterility and the amount of contamination present prior tosterilization. The irradiation may be conducted with the complex or thepharmaceutical composition maintained at from about 0° C. to about 30°C., e.g., from about 5° C. to about 20° C. or about about 10° C. toabout 15° C.

In some embodiments, a suitable dose of sterilizing radiation is a doseof about 10 kGy to about 25 kGy, e.g., about 15 kGy to about 20 kGy.

In some embodiments, when stored at 2° C., 8° C., or 25° C., thecomposition maintains a purity of at least 90% or greater (e.g., atleast 95% or greater) for a period of at least 24 hours followingexposure to gamma irradiation at a dose of about 10 kGy to about 25 kGy,e.g., about 15 kGy to about 20 kGy. For example, the period may be 3months, 6 months, 1 year, or 2 years. In some embodiments, a purity ofat least 90% or greater (e.g., 95% or greater) is maintained for aperiod of at least one month, e.g., following exposure to gammairradiation at a dose of about 10 kGy to about 25 kGy, e.g., about 15kGy to about 20 kGy. For example, the period may be from about one monthto about two months, from about two months to about three months, fromabout three months to about four months, from about four months to aboutfive months, from about five months to about six months, from about sixmonths to about one year, or from about one year to about two years.

Purity may be determined, for example, based on Reverse Phase HighPressure Liquid Chromatographic (RPLC) analysis of the composition. Forexample, RPLC spectra for the active agent in the irradiated compositioncan be compared with RPLC spectra for a USP standard of the activeagent. Peak retention times for the active agent in the irradiatedcomposition can be matched to the USP standard for the active agent, andimpurity peaks can be subtracted to obtain % purity levels.

Kits

A variety of kits may be provided which include one or more componentsof the pharmaceutical compositions disclosed herein along withinstructions for preparing and/or using the same. For example, in oneembodiment, a suitable kit may include a vehicle as described herein ina first container and a complex comprising pharmaceutically active agentas described herein in a second container, e.g., in powder form. Thesecomponents may then be mixed together prior to injection to form apharmaceutical composition according to the present disclosure. In someembodiments, the first container is a syringe which may be coupled tothe second container, e.g., a vial with a luer lock, to provide amechanism for mixing the vehicle and the complex comprisingpharmaceutically active agent. In other embodiments, both the first andsecond containers are syringes which may be coupled, e.g., via a luerlock, to provide a mechanism for mixing the vehicle and the complexcomprising pharmaceutically active agent.

In another embodiment, the pharmaceutical composition may be providedpre-mixed in a single container, e.g., a single syringe.

In another embodiment, the pharmaceutical composition may be providedun-mixed in a pre-filled, dual-chamber syringe including a first chambercontaining the vehicle and a second chamber containing the complexcomprising pharmaceutically active agent. The syringe may be providedsuch that a user can initiate contact and subsequent mixing of thevehicle and the complex comprising pharmaceutically active agent.

The instructions for use of the kit and/or kit components may beprovided as complete written instructions along with the kit, e.g., asan insert card or printed on the kit packaging; or stored on a computerreadable memory device provided with the kit. Alternatively, the kit mayinclude instructions which provide a brief instruction to the user anddirect the user to an alternate source for more complete useinstructions. For example, the kit may include a reference to aninternet site where the complete instructions for use may be accessedand/or downloaded.

Tissue Engineering and Medical Devices

In addition to pharmaceutical compositions, the complexes of the presentinvention may be used for tissue engineering and medical devices.Possible functional groups used for this application include PEGs,peptides, amino acids, amines, guanidiniums, PEGs, etc. For instance,scaffolds made from PCLs modified with the functional groups describedabove could be of great benefit by imparting greater hydrophilicity andbiocompatibility. Active pharmaceutical substances attached as prodrugsto the polymer chains could provide enhanced performance.

By functionalizing the polymers along the length of the chain, multiplepotential interaction sites are created, as opposed to only a few withtraditional polymers. For instance, precursor polymers (homopolymers orcopolymers) may be synthesized with pendant azide or alkyne groups, andthen these precursor polymers may be functionalized withalkyne-containing or azide-containing substrates using “click”chemistry.

The compositions disclosed herein may find use as coatings for medicaldevices, e.g., implantable medical devices. Such coatings may beapplied, e.g., by dip-coating the medical device prior to implantation.

As noted above, the compositions disclosed herein may find use asbiomaterials in tissue engineering. Tissue scaffolds and other usefulstructures may be fabricated by various methods including melt spinning,solvent spinning, electrospinning, and 3-D printing. Incorporation ofvarious functional groups could render scaffolds and other structuresmore hydrophilic, adhesive, and more biocompatible than materials thatare currently available.

Precursor Polymer Synthesis

In certain embodiments, precursor polymers according to the presentdisclosure are synthesized by an alcohol-initiated polymerizationreaction. For example, monomer starting material (e.g.,α-chloro-ε-caprolactone, ε-caprolactone, and the like) may be mixed withan alcohol, e.g., a mono or a poly-functional alcohol, such as but notlimited to, 1-dodecanol, 1,6-hexanediol, and the like. In someinstances, a catalyst is included in the polymerization reaction.Catalysts suitable for precursor polymer synthesis according toembodiments of the present disclosure include, but are not limited to,stannous 2-ethylhexanoate, stannic chloride dihydrate, and the like.Other catalysts may also be used, such as Lewis acids, alkyl metals, andorganic acids. In certain cases, the catalyst is included in thereaction in an amount ranging from 0.01 wt % to 5 wt %, such as 0.05 wt% to 3 wt %, including 0.1 wt % to 1 wt %. In some instances, thecatalyst is included in the reaction in an amount of 0.1 wt %. Incertain embodiments, the polymerization reaction is heated to atemperature of 100° C. or more, such as a temperature ranging from 100°C. to 200° C., or from 110° C. to 175° C., or from 120° C. to 150° C. Insome instances, the polymerization reaction is heated to a temperatureof 130° C. In certain embodiments, the polymerization reaction isallowed to proceed for a time period of overnight or longer. Forexample, the polymerization reaction may be allowed to proceed for 12hours or more, such as 18 hours or more, or 24 hours or more, or 30hours or more, or 36 hours or more, or 42 hours or more, or 48 hours ormore.

Examples of precursor polymer synthesis according to the presentdisclosure are described below in, e.g., Example 1.

Functionalizing Polymers

In certain embodiments, precursor polymers as described above may befunctionalized, i.e. their functionalizable side groups may betransformed into ionizable side groups to thereby provide thefunctionalized polymers described herein. Functionalization of theprecursor polymers may be performed using any of the various methods andtechniques known and available to those skilled in the art. For example,click chemistry reactions may be used to functionalize the precursorpolymers. In certain instances, a Huisgen 1,3-dipolar cycloadditionreaction (e.g., an azide-alkyne Huisgen cycloaddition reaction) may beused to functionalize the precursor polymers. In some instances, theprecursor polymers as described above may include a leaving group, suchas a halogen (e.g., chloro, bromo, iodo), a tosyl leaving group, and thelike. In some instances, the leaving group may be displaced by an azidemoiety (e.g., sodium azide) to form an azido substituted precursorpolymer, which may then undergo a click chemistry reaction with analkyne, such as in a Huisgen 1,3-dipolar cycloaddition reaction asdescribed above. In certain embodiments, a catalyst may be included inthe cycloaddition reaction. Catalysts suitable for cycloadditionreactions according to embodiments of the present disclosure include,but are not limited to, copper catalysts, such as copper iodide, and thelike. In certain cases, the catalyst is included in the reaction in anamount ranging from 0.01 equiv. to 1 equiv., such as 0.05 equiv. to 0.5equiv., including 0.1 equiv. to 0.3 equiv. In some instances, thecatalyst is included in the reaction in an amount of 0.1 equiv.

The functionalizing typically involves a solvent in which the precursorpolymer is soluble. In some embodiments, the solvent used during thefunctionalizing may comprise at least one member selected from an amide,cyclic amide, chlorinated hydrocarbon, ketone, ether, cyclic ether,polar aprotic, or protic solvent. Such solvents could include, but arenot limited to, DMF, NMP, chloroform, dichloromethane (DCM), acetone,tetrahydrofuran (THF), acetonitrile, dimethylsulfoxide (DMSO), water,and combinations thereof.

In many embodiments, the functionalizing comprises an exothermicreaction. In many embodiments, the functionalizing involves cooling andmixing. In some embodiments, the temperature of the functionalizingranges from 10° C. to 40° C., such as 20° C. to 30° C.

The purification may comprise dialysis, stirred cell purification,and/or tangential flow filtration. The pH of the purification typicallyfalls within the range of 3 to 10. For instance, purification ofamine-containing functionalized polymers often ranges from 4 to 5. Thepurification of acid-containing functionalized polymers often rangesfrom 8 to 9.

Examples of reactions for functionalizing polymers according to thepresent disclosure are described below in, e.g., Example 2.

In one specific aspect, there is provided a method comprising: providinga precursor polymer comprising repeat units, the repeat units comprisingfunctionalizable repeat units comprising at least one functionalizableside group; obtaining a functionalized polymer by transforming, usingclick chemistry, said functionalizable repeat units into ionizablerepeat units comprising at least one ionizable side group; and combiningthe functionalized polymer with a pharmaceutically active agent to forma complex in which a plurality of the at least one ionizable side groupsform a plurality of non-covalent bonds with the pharmaceutically activeagent. The complex may be a complex as defined elsewhere herein.

In this aspect, the transforming, using click chemistry, may compriseeffecting a cycloaddition reaction. The cycloaddition reaction may be aDiels-Alder cycloaddition reaction. The cycloaddition reaction may be aHuisgen 1,3-dipolar cycloaddition reaction. The cycloaddition reactionmay be a cycloaddition reaction between an azide and an alkyne to form alinkage comprising a 1,2,3-triazole.

The functionalizable side group may be an azido group and thetransforming, using click chemistry, may comprise reacting the precursorpolymer with an alkyne to form the functionalized polymer, thefunctionalized polymer comprising at least one 1,2,3-triazole ring.Alternatively, the functionalizable side group may be a leaving groupand the transforming, using click chemistry, may comprise: (a)transforming the leaving group into an azido group and thereby providinga polymer intermediate; and (b) reacting the polymer intermediate withan alkyne to form the functionalized polymer, the functionalized polymercomprising at least one 1,2,3-triazole ring. Examples of leaving groupinclude a halogen (e.g., chloro, bromo, iodo), a tosyl leaving group,and the like. In some embodiments the leaving group is a halogen, forexample chloro. The leaving group may be transformed into an azido groupby reacting the precursor polymer with sodium azide. The alkyne may be aterminal alkyne.

In some embodiments, the functionalizable side group may be an alkynylgroup and the transforming, using click chemistry, may comprise reactingthe precursor polymer with an azide to form the functionalized polymer,the functionalized polymer comprising at least one 1,2,3-triazole ring.The alkynyl group may, for example, be a terminal alkynyl group.

In some embodiments, the transforming, using click chemistry, comprisesa monovalent copper catalyzed reaction or a ruthenium catalyzedreaction. For example, the transforming, using click chemistry, maycomprise a monovalent copper catalyzed reaction, wherein a monovalentcopper catalyst is provided in the reaction through the ionization ofcopper iodide or copper bromide.

In some embodiments, the transforming, using click chemistry, comprisesa copper catalyzed azide-alkyne cycloaddition reaction. In someembodiments, the transforming, using click chemistry, occurs at leastpartially under degassing conditions.

In some embodiments, the precursor polymer comprises repeat units of theformula (I′):

wherein m′ is an integer from 1 to 10, and each R^(1′) and R^(2′) isindependently selected from hydrogen, hydroxyl, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted carbocyclyl,optionally substituted aryl, optionally substituted heterocyclyl,optionally substituted heteroaryl, a ionizable side group, and afunctionalizable side group. Such precursor polymers can readily beobtained by polymerization of monomers, such as monomers that are or arederivatives of caprolactone, lactic acid, glycolic acid, lactide,glycolide, butyrolactone, and valerolactone.

In some embodiments, the precursor polymer comprises functionalizablerepeat units of the formula (I′) wherein at least one of the R^(1′) sand R²′s is a functionalizable side group. In some embodiments, theprecursor polymer comprises repeat units of the formula (I′) whereineach R^(1′) and R^(2′) is independently selected from hydrogen, C₁₋₅alkyl and a functionalizable side group. In some embodiments, theprecursor polymer comprises repeat units of the formula (I′) wherein m′is an integer from 1 to 5. In some embodiments, the precursor polymercomprises repeat units of the formula (I′) wherein one of the R¹′s andR²′s is a functionalizable side group and all of the remaining R¹′s andR²′s are not functionalizable side groups.

In some embodiments, the precursor polymer comprises repeat units of theformula (I′) wherein m′ is 5, one R^(1′) is a functionalizable sidegroup and all of the remaining R¹′s and R²′s are hydrogen. Alternativelyor additionally, the precursor polymer may comprise repeat units of theformula (I′) wherein m′ is 5 and all of the R¹′s and R²′s are hydrogen.

In some embodiments, the precursor polymer comprises repeat units of theformula (I′) wherein m′ is 1, R^(1′) is hydrogen and R^(2′) is hydrogen.In some embodiments, the precursor polymer comprises repeat units of theformula (I′) wherein m′ is 1, R^(1′) is methyl and R^(2′) is hydrogen.In some embodiments, the precursor polymer comprises repeat units of theformula (I′) wherein m′ is 2, the R^(1′) and R^(2′) alpha to thecarbonyl group in formula (I′) are each hydrogen, the R^(1′) beta to thecarbonyl group is methyl and the R^(2′) beta to the carbonyl group ishydrogen. In some embodiments, the precursor polymer comprises repeatunits of the formula (I′) wherein m is 3 and all of the R¹′s and R²′sare hydrogen. In some embodiments, the precursor polymer comprisesrepeat units of the formula (I′) wherein m′ is 4, and all of the R¹′sand R²′s are hydrogen. In some embodiments, the precursor polymercomprises repeat units of the formula (I′) wherein m is 3, the R^(1′)and R^(2′) alpha to the carbonyl group in formula (I′) are eachhydrogen, the R^(1′) and R^(2′) beta to the carbonyl group are eachhydrogen, and the R^(1′) gamma to the carbonyl group is methyl and theR^(2′) gamma to the carbonyl group is hydrogen.

In some embodiments, the functionalizable group is selected from aleaving group, an azido group or an alkynyl group.

In some embodiments, the precursor polymer is a homopolymer of repeatunits of formula (I′) wherein at least one of the R¹′s and R²′s is afunctionalizable side group. In other embodiments, the precursor polymeris a copolymer comprising at least two different repeat units. Forexample the precursor polymer may be a copolymer wherein each of said atleast two different repeat units is of formula (I′) and wherein at leastone of said at least two different repeat units has a formula (I′) inwhich at least one of the R¹s and R²′s is a functionalizable side group.

In some embodiments, synthesis of the functionalized polymers involvesclick chemistry. In other embodiments, the synthesis of functionalizedpolymers does not involve click chemistry. Thus, in some embodiments,the ionizable groups may be included in the functionalized polymer usingclick chemistry. Alternatively, the ionizable groups may be added vianon-click chemistry routes.

Copper Catalyzed Click Chemistry

Monovalent copper catalyzed “click chemistry” cycloaddition reactionsmay be used to functionalize polymers with ionizable side groups toprovide functionalized polymers capable of forming complexes withpharmaceutically active agents as described herein. In some embodiments,ionizable side groups are bonded to precursor polymers via a monovalentcopper catalyzed azide-alkyne cycloaddition reaction. The monovalentcopper catalyst may be provided in the reaction through the ionizationof copper iodide or copper bromide.

Other Metal Catalysts

Cu is not the only metal capable of catalyzing this type ofcycloaddition. As long as the metal is or can become coordinativelyunsaturated, other metals known to form stable acetylides may also beemployed. Exemplary metals that can form stable acetylides include Cu,Au, Ag, Hg, Cd, Zr, Ru, Fe, Co, Pt, Pd, Ni, Rh, and W. It is a matter offinding the right metal/ligand combination.

Catalysis of Ligation Reaction by Metallic Container: Metalliccontainers can also be used as a source of the catalytic species tocatalyze the ligation reaction. For example, a copper container (Cu⁰)may be employed to catalyze the reaction. In order to supply thenecessary ions, the reaction solution must make physical contact withthe copper surface of the container. Alternatively, the reaction may berun in a non-metallic container, and the catalytic metal ions suppliedby contacting the reaction solution with a copper wire, copper shavings,or other structures. Although these reactions may take longer to proceedto completion, the experimental procedure is relatively simple.

Alternative Reducing Agents: Metals may be employed as reducing agentsto maintain the oxidation state of the Cu (I) catalyst or of other metalcatalysts. Preferred metallic reducing agents include Cu, Al, Be, Co,Cr, Fe, Mg, Mn, Ni, and Zn. Alternatively, an applied electric potentialmay be employed to maintain the oxidation state of the catalyst.

Cu(I) Salt Used Directly: If Cu(I) salt is used directly, no reducingagent is necessary, but acetonitrile or one of the other ligandsindicated above should be used as a solvent (to prevent rapid oxidationof Cu(I) to Cu(II) and one equivalent of an amine should be added (toaccelerate the otherwise extremely slow reaction-vide supra). In thiscase, for better yields and product purity, oxygen should be excluded.Therefore, the ascorbate (or any other reducing) procedure is oftenpreferred over the unreduced procedure. The use of a reducing agent isprocedurally simple, and furnishes triazole products in excellent yieldsand of high purity. For instance, in some cases, the yield of thefunctionalizing step may range from 40% to 90%, such as 45% to 80% or50% to 75%. In the exemplary click chemistry synthesis route shownbelow, an azide (—N₃) is placed on the polymer chain and functionalizedvia click reaction. It may be accomplished in an advantageous “one pot”approach. It is also advantageous in that it negates the need forsynthesizing small molecule azides that are potentially explosive.

Another exemplary click chemistry reaction scheme is shown below:

In the above reaction scheme, Baeyer-Villiger oxidation was performed onα-chlorocyclohexanone. The crude reaction product may be purified togive desired monomer.

Also in the above reaction scheme, copolymer was reacted with the“click” reagents to form the desired amine-functionalized complexationpolymer.

The click reaction is typically followed by purification. Purificationmay involve dialysis, stirred cell, ultrafiltration, and/or tangentialflow filtration. The purification may be conducted at various pH, suchas from 4 to 9, such as 4 to 5 for amine functionalized polymers, andsuch as 8 to 9 for acid functionalized polymers.

Polymer Synthesis via Non-Click Chemistry

The below exemplary reaction scheme, which does not involve clickchemistry, has been used to form a functionalized polymer, i.e., anamine-terminated polymer.

In some embodiments, a functionalized polymer according to the presentdisclosure, which is not produced via click chemistry, comprises atleast one terminal repeat unit that has the formula (I″).

wherein m″ is an integer from 1 to 10, each R¹″ and R²″ is independentlyselected from hydrogen, hydroxyl, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted carbocyclyl,optionally substituted aryl, optionally substituted heterocyclyl,optionally substituted heteroaryl, and a ionizable side group; and R³ isan ionizable end group.

By “terminal repeat unit” is meant a repeat unit that is at the end ofthe polymer chain. In some embodiments a repeat unit of the formula (I″)is present at both ends of the polymer chain. In some embodiments arepeat unit of the formula (I″) is present at one end of the polymerchain.

In some embodiments, the chemical identities and combinations of thegroups m″ and R¹″ and R²″ are as described herein with reference to theformula (I) (i.e., with reference to the groups m and R¹ and R²). Insome embodiments, the ionizable end group R³ comprises at least onemember selected from ammonium, carboxylate, hydrazinium, guanidinium,sulfate, sulfonate, phosphonate and phosphate. Methods for attachingchemical end groups, including ionizable end groups, to polymers arewell known in the art and can be adopted in order to prepare theabove-described polymers.

In some embodiments, these functionalized, ionizable end groupcomprising polymers find use in the formation of complexes withrelatively low molecular weight pharmaceutically active agents, e.g.,pharmaceutical active agents having a weight average molecular weight ornumber average molecular weight ranging from 300 Daltons to 2,500Daltons, e.g., from 500 Daltons to 2,000 Daltons, or from 1,000 Daltonsto 1,500 Daltons, as determined using gel permeation chromatography orNMR spectra respectively.

Further examples of functionalized polymers made through non-clickchemistry techniques include, but are not limited to, poly(L-serine) orpoly (L-serine ester) (JACS 1992, 114, 3962-3965; Macromolecules 1990,23, 3399-3406; Polymer Bulletin 1990, 24, 349-353); synthetic poly(aminoacids) (Kohn, Biodegradable Polymers as Drug Delivery Systems, 1990,195-229); polycondensation of serinol (J Control Release 2005, 101,21-34); poly(vinylpyridine) (Sigma Aldrich Part #472352); and poly(vinylamine) analogs (PCT Publication No. WO 1998/018833).

Preparation of Complexes

In certain embodiments, functionalized polymers as described above mayform a complex with a pharmaceutically active agent. In some instances,the pharmaceutically active agent may be dissolved in a solution.Suitable solvents for dissolving the pharmaceutically active agentinclude solvents in which the pharmaceutically active agent is soluble.In some cases, the solvent is compatible with the pharmaceuticallyactive agent, such that the solvent is non-reactive with thepharmaceutically active agent and does not cause significant degradationof the pharmaceutically active agent or the product complex. Examples ofsuitable solvents include water, which may also include a buffer (e.g.,NaHCO₃). In some instances, the functionalized polymer may be dissolvedin a solution. Suitable solvents for dissolving the functionalizedpolymer include solvents in which the functionalized polymer is soluble.In some cases, the solvent is compatible with the functionalizedpolymer, such that the solvent is non-reactive with the functionalizedpolymer and does not cause significant degradation of the functionalizedpolymer or the product complex. Examples of suitable solvents includewater, which may also include a buffer (e.g., NaHCO₃), DMSO, NMP, ethylacetate, and benzyl alcohol.

In certain embodiments, a solution of the functionalized polymer and thepharmaceutically active agent may be combined to form a complex betweenthe functionalized polymer and the pharmaceutically active agent. Insome instances, the complex between the functionalized polymer and thepharmaceutically active agent is less soluble in the complexationsolvent than the functionalized polymer and/or the pharmaceuticallyactive agent themselves. The term “complexation solvent” is used hereinto differentiate between the solvent or solvents used in thecomplexation of the functionalized polymer with the pharmaceuticallyactive agent and other solvents described herein, e.g., solventsutilized in a vehicle component of a composition. For instance, thecomplex between the functionalized polymer and the pharmaceuticallyactive agent may be substantially insoluble in the complexation solventand form an isolatable precipitate. Isolation of the complex (e.g.,separation of the precipitate from the supernatant) may be performed byany convenient isolation method, such as centrifugation, filtration,etc. In certain instances, the isolated complex is dried to form a driedcomposition of the complexed pharmaceutically active agent. For example,the isolated complex may be dried by any convenient drying method, suchas, but not limited to, spray drying, freeze drying (i.e.,lyophilization), drying under reduced atmospheric pressure (e.g., dryingunder vacuum), thin film evaporation, combinations thereof, and thelike. In certain instances, the complex is a spray-dried complex. Incertain instances, the complex is a lyophilized complex.

In some embodiments, the formation of the complex occurs at atemperature ranging from 10° C. to 40° C., such as 20° C. to 30° C.

Examples of the preparation of complexes according to the presentdisclosure are described below in, e.g., Example 3.

The present invention will be further illustrated by way of thefollowing Examples. These examples are non-limiting and do not restrictthe scope of the invention. Unless stated otherwise, all percentages,parts, etc. presented in the examples are by weight.

EXAMPLES Example 1: Examples of Precursor Polymer Synthesis

The following are examples of precursor polymer synthesis. The resultingprecursor polymers are then used to form complexing polymers, e.g., asdescribed in Example 2.

Example 1A

In a 40 mL-screw top vial, 29.73 g of α-chloro-ε-caprolactone (madeaccording to R. Jerome, Macromolecules, vol. 37, 2004, p. 4055), and0.28 grams 1-dodecanol were mixed at ambient temperature. Stannous2-ethylhexanoate was added as a solution in dry toluene. The amount ofcatalyst added was approximately 0.1 wt %. The vial was then placed in ablock heater at 130° C. and agitated by hand periodically. Thepolymerization was allowed to proceed for 19.5 hours, and the vial wasremoved from the heating block. The resulting polymer had a numberaverage molecular weight (Mn) of 20.8 kDa as measured by ¹H NMR and aweight average molecular weight (Mw) of 30.5 kDa as measured by GPC inTHF. Similar lots were prepared with varying amounts of 1-dodecanol toprovide polymers having an Mn by NMR of 25.2 kDa and a Mw by GPC of 29.2kDa, an Mn by NMR of 22.0 kDa and a Mw by GPC of 41.9 kDa, an Mn by NMRof 14.0 kDa and a Mw by GPC of 20.6 kDa, and an Mn by NMR of 24.0 kDaand a Mw by GPC of 27.8 kDa.

Example 1B

In a 20 mL-screw top vial, 4.95 g of α-chloro-ε-caprolactone, 4.95 g ofDL-lactide, and 0.09 grams 1-dodecanol were mixed at ambienttemperature. Stannous 2-ethylhexanoate was added as a solution in drytoluene. The amount of catalyst added was approximately 0.1 wt %. Thevial was then placed in a block heater at 130° C. and agitated by handperiodically. The polymerization was allowed to proceed for 19.5 hours,and the vial was removed from the heating block. The resulting polymerhad a number average molecular weight (Mn) of 18.0 kDa as measured by ¹HNMR and a weight average molecular weight (Mw) of 32.9 kDa as measuredby GPC in THF. The molar ratio of α-chloro-ε-caprolactone to DL-lactidewas 50:50. Similar lots were prepared with varying ratios of monomersand amounts of 1-dodecanol to provide polymers having weight averagemolecular weight (Mw) of 5.6 and 31.8 kDa.

Example 1C

The procedure in Example 1B was repeated except that the components ofthe synthesis were: ε-caprolactone (4.95 grams), α-chloro-ε-caprolactone(4.96 grams), and 1-dodecanol (0.09 grams). The resulting polymer had anumber average molecular weight (Mn) of 17.2 kDa as measured by ¹H NMRand a Mw by GPC of 42.7 kDa. The molar ratio of α-chloro-ε-caprolactoneto ε-caprolactone was 44:56. Similar lots were prepared with varyingamounts of 1-dodecanol to provide polymers having weight averagemolecular weight (Mw) of 5.4 kDa, 5.6 kDa, 11.4 kDa, 12 kDa, 15.9 kDa,16.0 kDa, and 16.8 kDa.

Example 1D

The procedure in Example 1B was repeated except that the components ofthe synthesis were: glycolide (3.40 grams), α-chloro-ε-caprolactone(6.52 grams), and 1-dodecanol (0.09 grams). The resulting polymer had anumber average molecular weight (Mn) of 20.0 kDa as measured by ¹H NMR.The molar ratio of a-chloro-ε-caprolactone to glycolide was 60:40.

Example 1E

ε-Caprolactone (62.97 grams), α-chloro-ε-caprolactone (62.98 grams), and1-dodecanol (4.02 grams) were added to a 250-m L, 3-neck round bottomflask. The flask was sealed with a glass stopper, a gas joint with astopcock, and a stirrer bearing with a glass shaft and Teflon® paddle.The ambient atmosphere was removed from the flask under vacuum, and theflask was back-filled with nitrogen gas. The flask was placed in an oilbatch at 130° C. and stirred under a positive pressure of nitrogen gas.After 30 minutes, stannous 2-ethylhexanoate was added as a solution indry toluene. The amount of catalyst added was approximately 0.05 wt %.The polymerization was allowed to proceed for 29 hours. Next, the solidpolymer was subjected to vacuum to remove residual monomer for 30minutes. Then the contents of the flask were discharged from the flaskinto glass containers and allowed to cool. The total yield of polymerwas 116.9 g, 90%. The resulting polymer had a number average (Mn)molecular weight of 5.3 kDa as measured by ¹H NMR and a weight averagemolecular weight (Mw) of 15.7 kDa by GPC in THF. The molar ratio ofα-chloro-ε-caprolactone to ε-caprolactone was 43:57.

Example 1F

The procedure in Example 1E was repeated except that the components ofthe synthesis were: ε-Caprolactone (64.40 grams),α-chloro-ε-caprolactone (64.41 grams), and 1-dodecanol (1.21 grams). Thepolymerization was allowed to proceed for 40 hours. Next, the solidpolymer was subjected to vacuum to remove residual monomer for 30minutes. Then the contents of the flask were discharged from the flaskinto glass containers and allowed to cool. The total yield of polymerwas 107.88 g, 83%. The resulting polymer had a number average molecularweight (Mn) of 17.0 kDa as measured by ¹H NMR and a weight averagemolecular weight (Mw) of 44.0 kDa by GPC in THF. The molar ratio ofα-chloro-ε-caprolactone to ε-caprolactone was 44:56.

Example 1G

In a 40 mL-screw top vial, 7.31 g of α-chloro-ε-caprolactone, 17.5 g ofDL-lactide, 4.38 g of glycolide, and 0.08 g of 1-dodecanol were mixed atambient temperature. The catalyst stannous 2-ethylhexanoate was added asa solution in dry toluene. The amount of catalyst added wasapproximately 0.1 wt %. The vial was then placed in a block heater at130° C. and agitated by hand periodically. The polymerization wasallowed to proceed for 42 hours, and the vial was removed from theheating block. The resulting polymer had a number average molecularweight (Mn) of 5.1 kDa as measured by ¹H NMR and a weight averagemolecular weight (Mw) of 17.5 kDa as measured by GPC in THF. The molarratio of α-chloro-ε-caprolactone to DL-lactide to glycolide was26:56:18.

Example 2: Synthesis of Complexation Polymers

The following examples involve converting precursor polymers, such asthose formed in above Example 1, to form functionalized complexationpolymers.

Example 2A Amine Functionalized Copolymer

The precursor polymers utilized in this Example were prepared asdescribed above in Examples 1E and 1F.

To a solution of the precursor α-chloro-ε-caprolactone-co-ε-caprolactonecopolymer in N,N-dimethylformamide (DMF; 4 mL solvent/g polymer) wasadded sodium azide (NaN₃, 1.2 equiv.) with vigorous stirring via amagnetic stir bar. The flask was evacuated and back-filled with nitrogenfive times and was stirred overnight at room temperature. The presenceof a new organic azide moiety was confirmed by FT-IR (at ˜2100 cm⁻¹).The flask was cooled to 0° C. in an ice bath and was evacuated andback-filled with nitrogen five times. Under a stream of nitrogen,propargyl amine (1.0 equiv.), triethylamine (0.1 equiv.), and copperiodide (0.1 equiv.) were added sequentially. The flask was againevacuated and back-filled with nitrogen. The exothermic reaction wasstirred at 0° C. for an hour; the ice bath was then removed, and thereaction was stirred vigorously overnight under nitrogen. FT-IRconfirmed the disappearance of the organic azide at ˜2100 cm⁻¹,indicating the reaction was complete. The solids were filtered out ofthe reaction mixture and were washed with ample DMF. The filtrate wasdiluted with 0.01N hydrochloric acid until the solution was homogeneousand the pH was 4-5. A metal scavenger (SiliaMetS® Thiol, 8 equiv.) wasadded to the polymer solution, and the mixture was stirred overnight.The scavenger was filtered out, and the resulting filtrate was purifiedvia ultrafiltration in a stirred cell using 1.5 diavolumes of purifiedwater as the dialysate. The purified polymer solution was thentransferred to VirTis® jars, shell-frozen, and lyophilized until dry.The resulting functionalized complexation polymers were called AFCP-1(prepared using 15.7 kDa precursor polymer described in Example 1E) andAFCP-2 (prepared using 44.0 kDa precursor polymer described in Example1F). A chemical formula for the amine functionalized complexationpolymers produced in this example is provided below.

Example 2B: Amine Functionalized Homopolymer

The precursor polymer utilized in this Example was the 29.2 kDa polymerprepared as described above in ExamplelA.

To a solution of the precursor poly(α-chloro-ε-caprolactone) inN-methylpyrrolidone (NMP; 5 mL solvent/g polymer) was added sodium azide(NaN₃, 1.2 equiv.) with vigorous stirring via a magnetic stir bar. Theflask was evacuated and back-filled with nitrogen five times and wasstirred overnight at room temperature. The presence of a new organicazide moiety was confirmed by FT-IR (at ˜2100 cm⁻¹). The flask wascooled to 0° C. in an ice bath and was evacuated and back-filled withnitrogen five times. Under a stream of nitrogen, propargyl amine (1.0equiv.), triethylamine (0.1 equiv.), and copper iodide (0.1 equiv.) wereadded sequentially. The flask was again evacuated and back-filled withnitrogen. The exothermic reaction stirred at 0° C. for an hour; the icebath was then removed, and the reaction continued to stir vigorouslyovernight under nitrogen. FT-IR confirmed the disappearance of theorganic azide at ˜2100 cm⁻¹, indicating the reaction was complete. Thesolids were filtered out of the reaction mixture and were washed withample NMP. The filtrate was diluted with 0.01N hydrochloric acid untilthe solution was homogeneous and the pH was 4-5. A metal scavenger(SiliaMetS® Thiol, 8 equiv.) was added to the polymer solution, and themixture was stirred overnight. The scavenger was filtered out, and theresulting filtrate was purified via ultrafiltration in a stirred cellusing 1.5 diavolumes of purified water as the dialysate. The purifiedpolymer solution was then transferred to VirTis® jars, shell-frozen, andlyophilized until dry.

Example 2C: Amine Functionalized Copolymer

The precursor polymers utilized in this Example were the 5.4 kDa, 11.4kDa, and 16.8 kDa polymers prepared as described above in Example 1C.The procedure from Example 2A was followed, but the polymer solution waspurified with conventional dialysis membrane tubing (Spectra/Por® MWCO=1kDa or 3.5 kDa).

Example 2D: Amine Functionalized Copolymer

The precursor polymer utilized in this Example was the 15.7 kDa polymerprepared as described above in Example 1E. The procedure from Example 2Bwas followed, but the polymer solution was purified with a tangentialflow filtration (TFF) system using deionized water as the dialysate.

Example 2E: Carboxylate Functionalized Copolymer

The precursor polymers utilized in this Example were the 5.6 kDa, 12 kDaand the 15.9 kDa polymers prepared as described above in Example 1C.

To a solution of the precursor α-chloro-ε-caprolactone-co-ε-caprolactonecopolymer in N,N-dimethylformamide (DMF; 4 mL solvent/g polymer) wasadded sodium azide (NaN₃, 1.2 equiv.) with vigorous stirring via amagnetic stir bar. The flask was evacuated and back-filled with nitrogenfive times and was stirred overnight at room temperature. The presenceof a new organic azide moiety was confirmed by FT-IR (at ˜2100 cm⁻¹).The flask was cooled to 0° C. in an ice bath and was evacuated andback-filled with nitrogen five times. Under a stream of nitrogen,5-hexynoic acid (1.0 equiv.), triethylamine (0.1 equiv.), and copperiodide (0.1 equiv.) were added sequentially. The flask was againevacuated and back-filled with nitrogen. The exothermic reaction wasstirred at 0° C. for an hour; the ice bath was then removed, and thereaction was stirred vigorously overnight under nitrogen. FT-IRconfirmed the disappearance of the organic azide moiety, indicating thereaction was complete. The solids were filtered out of the reactionmixture and were washed with ample DMF. The filtrate was diluted withsaturated aqueous sodium bicarbonate solution until the solution washomogeneous and the pH was 8-9. DOWEX™ Marathon™ C sulfonic acid ionexchange resin was washed with ample methanol to remove all color, thenadded to the polymer solution, and the mixture was stirred overnight.The resin was filtered out, and the resulting filtrate was purified viadialysis against deionized water using Spectra/Por® dialysis tubingovernight. The purified polymer solution was then transferred to VirTis®jars, shell-frozen, and lyophilized until dry. The carboxylatefunctionalized copolymers prepared with the 5.6 kDa, 12 kDa and 15.9 kDaprecursor polymers correspond to the CFCP-3, CFCP-2, and CFCP-1copolymers, respectively.

Example 2F: Guanidinium Functionalized Copolymer

The precursor polymers utilized in this Example were the 5.4 kDa, 11.4kDa, and 16.8 kDa polymers prepared as described above in Example 1C.

To a solution of the precursor α-chloro-ε-caprolactone-co-ε-caprolactonecopolymer in N,N-dimethylformamide (DMF; 4 mL solvent/g polymer) wasadded sodium azide (NaN₃, 1.2 equiv.) with vigorous stirring via amagnetic stir bar. The flask was evacuated and back-filled with nitrogenfive times and was stirred overnight at room temperature. The presenceof a new organic azide moiety was confirmed by FT-IR (at ˜2100 cm⁻¹).The flask was cooled to 0° C. in an ice bath and was evacuated andback-filled with nitrogen five times. Under a stream of nitrogen, thepropargyl amide of arginine dihydrochloride (structure shown below; 1.0equiv.), triethylamine (10 equiv.), and copper iodide (0.1 equiv.) wereadded sequentially.

The flask was again evacuated and back-filled with nitrogen. Theexothermic reaction was stirred at 0° C. for an hour; the ice bath wasthen removed, and the reaction was stirred vigorously overnight undernitrogen. FT-IR confirmed the disappearance of the organic azide moiety,indicating the reaction was complete. The solids were filtered out ofthe reaction mixture and were washed with ample DMF. The filtrate wasdiluted with 0.01N HCl until the solution was homogeneous and the pH was3-4. The resulting solution was purified via dialysis using Spectra/Por®dialysis membrane tubing with MWCO=100-500 Da against 0.01N HClovernight, then with MWCO=1000 Da tubing for one hour. The purifiedpolymer solution was then transferred to VirTis® jars, shell-frozen, andlyophilized until dry.

Example 2G: Amine Functionalized Homopolymer

The precursor polymer utilized in this Example was the 41.9 kDa polymerprepared according to the method described in Example 1A above.

A solution of the precursor poly(α-chloro-ε-caprolactone) in DMF (4 mLsolvent/g polymer) was evacuated and back-filled with nitrogen fivetimes. Sodium azide (NaN₃, 1.2 equiv.) was added and stirred overnightat room temperature via a magnetic stir bar. The presence of a neworganic azide moiety was confirmed by FT-IR (at 2106 cm⁻¹). Under astream of nitrogen, propargyl amine (1.0 equiv.) and triethylamine (0.1equiv.) were added sequentially. The flask was again evacuated andback-filled with nitrogen 5 times then copper iodide (0.1 equiv.) wasadded, followed by one additional evacuate/back-fill cycle. The reactionimmediately became hot and was placed in an ice bath to control theexotherm. After 2 hours, the ice bath was removed, and the reaction wasstirred overnight at room temperature under nitrogen. FT-IR confirmedthe disappearance of the organic azide peak at ˜2106 cm⁻¹, indicatingthe reaction was complete. The contents of the flask were centrifuged,and the green supernatant was decanted off; the solid bed was washedwith ample DMF, centrifuged again, and the supernatants combined. TheDMF was largely removed via rotary evaporation, and the thick green oilwas dissolved in 0.01 N HCl over many hours. This solution was dialyzedin MWCO=3500 Spectra/Por® dialysis tubing against 0.01 N HCl for twodays, changing the dialysate 4 times. The polymer solution was thenfreeze-dried for three days until dry.

Example 2H: Amine Functionalized Copolymer

The precursor polymer utilized in this Example was the 16.0 kDa polymerprepared according to the method described in Example 1C above.

A solution of the precursorpoly(α-chloro-ε-caprolactone-co-ε-caprolactone) in DMF (4 mL solvent/gpolymer) was evacuated and back-filled with nitrogen five times. Sodiumazide (NaN₃, 1.2 equiv.) was added and stirred overnight at roomtemperature via a magnetic stir bar. The presence of a new organic azidemoiety was confirmed by FT-IR (at 2105 cm⁻¹). The flask was cooled to 0°C. in an ice bath and was evacuated and back-filled with nitrogen fivetimes. Under a stream of nitrogen, propargyl amine (1.0 equiv.) andsodium ascorbate (0.12 equiv.) were added sequentially. The flask wasagain evacuated and back-filled with nitrogen 5 times, then coppersulfate (0.05 equiv.) was added, followed by one additionalevacuate/back-fill cycle. The reaction was stirred at 0° C. for 1 hour,then overnight at room temperature under nitrogen. FT-IR confirmed thedisappearance of the organic azide peak at ˜2106 cm⁻¹, indicating thereaction was complete. The thick green polymer ball was then directlydissolved in 0.01 N HCl and dialyzed using Spectra/Por® regeneratedcellulose tubing with MWCO=3500 against 0.01 N HCl for two days,changing the dialysate 2 times. The polymer solution was thenlyophilized until dry.

Example 2I: Carboxylate Functionalized Homopolymer

The precursor polymer utilized in this example was the 30.5 kDa polymerprepared according to the method described in Example 1A above.

A solution of the precursor poly(α-chloro-ε-caprolactone) in DMF (4 mLsolvent/g polymer) was evacuated and back-filled with nitrogen fivetimes. Sodium azide (NaN₃, 1.2 equiv.) was added and stirred overnightat room temperature via a magnetic stir bar. The presence of a neworganic azide moiety was confirmed by FT-IR (at 2107 cm⁻¹). The flaskwas cooled to 0° C. in an ice bath and was evacuated and back-filledwith nitrogen five times. Under a stream of nitrogen, 5-hexynoic acid(1.2 equiv.) and triethylamine (0.1 equiv.) were added sequentially. Theflask was again evacuated and back-filled with nitrogen 5 times thencopper iodide (0.1 equiv.) was added, followed by one additionalevacuate/back-fill cycle. The reaction was stirred at 0° C. for 1 hour,then overnight at room temperature under nitrogen. FT-IR confirmed thedisappearance of the organic azide peak at ˜2106 cm⁻¹, indicating thereaction was complete. The contents of the flask were centrifuged, andthe green supernatant was decanted off; the solid bed was washed withample DMF, centrifuged again, and the supernatants combined. The DMF waslargely removed via rotary evaporation, and the thick green oil wasdissolved in a 12:1 mixture of water:triethylamine over many hours. Thebrownish green polymer solution was dialyzed using Spectra/Por®regenerated cellulose tubing with MWCO=3500 against deionized water fortwo days, changing the dialysate 3 times. The polymer solution was thenlyophilized until dry. The green polymer was then dissolved in methanoland stirred with Marathon™ C cation exchange resin for 3 hours. Theresin was filtered out, and the methanol was removed via rotaryevaporation. To the polymer residue was added purified water, causingthe polymer to precipitate. Ammonium hydroxide was added dropwise untilthe polymer went into solution; the final pH was 8. This solution wasdialyzed for two days using MWCO=1000 Spectra/Por® dialysis tubing withwater as the dialysate, replacing the dialysate 2 times. The polymersolution was then transferred to VirTis® jars, shell-frozen, andlyophilized until dry.

Example 2J: Guanidinium Functionalized Homopolymer

The precursor polymer utilized in this example was the 29.2 kDa polymerprepared according to the method described in Example 1A above.

To a solution of the precursor poly(α-chloro-ε-caprolactone) inN,N-dimethylformamide (DMF; 4 mL solvent/g polymer) was added sodiumazide (NaN₃, 1.2 equiv.) with vigorous shaking on an orbital shaker. Thevial was evacuated and back-filled with nitrogen five times and wasshaken overnight at room temperature. The presence of a new organicazide moiety was confirmed by FT-IR (at 2106 cm⁻¹). Under a stream ofnitrogen, the propargyl amide of arginine dihydrochloride andtriethylamine (3 equiv.) were added sequentially. The flask was againevacuated and back-filled with nitrogen five times, and copper iodide(0.1 equiv.) was added. The reaction shook overnight under nitrogen,with close temperature monitoring over the first hour. FT-IR confirmedthe disappearance of the organic azide moiety, indicating the reactionwas complete. The contents of the vial were directly dissolved in 0.01NHCl until the solution was homogeneous and the pH was 3-4. The resultingsolution was purified via dialysis using Spectra/Por® dialysis membranetubing with MWCO=1000 Da against 0.01N HCl over 2 nights, changing thedialysate twice. The purified polymer solution was then transferred to aVirTis® jar, shell-frozen, and lyophilized until dry.

Example 2K

The precursor polymer utilized in this example was a 27.8 kDa Mw polymerprepared according to the method described in Example 1A above.

A solution of intermediate poly(α-chloro-ε-caprolactone) in DMF (4 mLsolvent/g polymer) was evacuated and back-filled with nitrogen fivetimes. Sodium azide (NaN₃, 1.2 equiv.) was added and stirred overnightat room temperature via a magnetic stir bar. The presence of a neworganic azide moiety was confirmed by FT-IR (at 2106 cm⁻¹). Under astream of nitrogen, propargyl amine (1.0 equiv.) and sodium ascorbate(0.12 equiv.) were added sequentially. The vial was again evacuated andback-filled with nitrogen 5 times, then copper sulfate (0.05 equiv.) wasadded, followed by one additional evacuate/back-fill cycle. The reactionstirred overnight under nitrogen. FT-IR confirmed the disappearance ofthe organic azide peak at ˜2106 cm⁻¹, indicating the reaction wascomplete. The contents of the vial were centrifuged and the greensupernatant decanted off; the solid bed was washed with ample DMF,centrifuged again, and the two supernatants combined. The DMF waslargely removed via rotary evaporation, and the polymer was precipitatedinto water as a dark green semi-solid. The polymer was washed severaltimes with fresh water and dried under vacuum overnight. The polymer wasthen dissolved in a 1:1 mixture of acetone/water and dialyzed usingSpectra/Por® regenerated cellulose tubing with MWCO=1000 against 1:1acetone/water. The dialysate was replaced after 5 hours, and dialysiscontinued overnight. The dialysate was then replaced with fresh water,and within minutes, polymer began to precipitate within the membrane.The solution was drained from the dialysis tube, shell-frozen at −6° C.,and placed under vacuum. After 7 days, the polymer appeared to be dry,but was still dark green and insoluble in most common organic solvents.Assuming it was still impure, the polymer was dissolved in 0.01 N HCl,and the solution color began to change from dark green to amber almostimmediately. This solution was dialyzed in MWCO=3500 dialysis tubingagainst 0.01 N HCl for two days, changing the dialysate 4 times. Thepolymer solution was then freeze-dried for two days until dry.

Example 2L

The precursor polymer utilized in this example was the 44.0 kDa Mwpolymer prepared according to the method described in Example 1F above.

A solution of intermediatepoly(α-chloro-ε-caprolactone-co-ε-caprolactone) in DMF (4 mL solvent/gpolymer) was evacuated and back-filled with nitrogen five times. Sodiumazide (NaN₃, 1.2 equiv.) was added and stirred overnight at roomtemperature via a magnetic stir bar. The presence of a new organic azidemoiety was confirmed by FT-IR (at 2104 cm⁻¹). The flask was cooled to 0°C. in an ice bath and was evacuated and back-filled with nitrogen fivetimes. Under a stream of nitrogen, propargyl alcohol (1.0 equiv.) andtriethylamine (0.1 equiv.) were added sequentially. The flask was againevacuated and back-filled with nitrogen 5 times then copper iodide (0.1equiv.) was added, followed by one additional evacuate/back-fill cycle.The reaction stirred at 0° C. for 1 hour, then overnight at roomtemperature under nitrogen. FT-IR confirmed the disappearance of theorganic azide peak at ˜2104 cm⁻¹, indicating the reaction was complete.The contents of the flask were centrifuged and the green supernatantdecanted off; the solid bed was washed with ample DMF, centrifugedagain, and the supernatants were combined. The DMF was largely removedvia rotary evaporation, and the thick green oil was suspended in water.The flask was rotated in a warm water bath overnight, but the polymerdid not dissolve. The water was poured off, and dissolution wasattempted in chloroform, acetonitrile, tetrahydrofuran, and acetone.Each time, the polymer did not dissolve. The polymer eventuallydissolved in NMP, and the solution was transferred to a beaker andtreated with SiliaMetS® Thiol resin overnight. The resin was filteredout, and the polymer solution was transferred to Spectra/Por® dialysistubing with MWCO=3500. The material dialyzed against NMP for 5 hours.The dialysate was discarded and replaced with purified water anddialyzed for 2 days. At this point, much of the polymer had begun toprecipitate as a sticky substance and was leaking out of the dialysismembranes. What remained in the dialysis tubes was poured into a Virtisjar and lyophilized for 1 week until dry.

Example 2M

A solution of intermediate poly(α-chloro-ε-caprolactone) in THF (4 mLsolvent/g polymer) was evacuated and back-filled with nitrogen fivetimes. Sodium azide (NaN₃, 1.2 equiv.) was added and stirred overnightat room temperature via a magnetic stir bar, but no organic azide peak(2100 cm⁻¹) appeared. The reaction vial was transferred to a tumbler andtumbled to effect more efficient mixing, but still no azide peakappeared. The reaction was heated to 40° C. and shaken every hour byhand, but after 6 hours, no azide peak could be observed in the FT-IR.The reaction was discarded. While not intending to be bound by anyparticular theory, it was proposed that NaN₃ is not sufficiently solublein THF to react with the chlorinated polymer.

Example 2N

To a solution of intermediate poly(α-chloro-ε-caprolactone) in THF (4 mLsolvent/g polymer) was added purified water (10:1 THF:water). The vialwas evacuated and back-filled with nitrogen five times. Sodium azide(NaN₃, 1.2 equiv.) was added and stirred overnight at room temperaturevia a magnetic stir bar, but no organic azide peak (˜2100 cm⁻¹)appeared. The reaction vial was transferred to a tumbler and tumbled toeffect more efficient mixing, but still no azide peak appeared; thesodium azide clumped together with the water and was not free-flowing inthe reaction vial. The reaction was heated to 40° C. and shaken everyhour by hand, but after 6 hours, no azide peak could be observed in theFT-IR. While not intending to be bound by any particular theory, it wasproposed that the addition of more water to the reaction could haveincreased the solubility of sodium azide, however the polymer wouldlikely have precipitated under those conditions. Therefore, the reactionwas discarded.

Yields for Examples 2A to 2F ranged from 43% to 70%, depending on thenature of the “clicked” group and the method of purification.

Example 3: Complexation of Exenatide or GLP-1 Analog with FunctionalizedComplexation Polymers

Complexation between exenatide or GLP-1 analog and functionalizedcomplexation polymers was determined as set forth below.

Materials and Methods

Pharmaceutically active agents (exenatide and a GLP-1 analog) werecombined as set forth below with an amine-functionalized homopolymer, anamine-functionalized copolymer, a carboxylate-functionalizedhomopolymer, and a guanidinium-functionalized homopolymer.

Preparation of either lyophilized (exenatide) or spray dried (GLP-1analog) powder of complexed pharmaceutically active agents: 1.00 g ofbiologically active powder was placed in a 150 mL wide-mouth glass jar.100 mL of 50 mM NH₄HCO₃ solution were added, and the mixture was stirredat 400 rpm for 30 min at room temperature (until it became a visiblyclear solution).

100 mL of a 10 mg/mL solution of functionalized complexation polymerwere then prepared by stirring 1.00 g of polymer in 100 mL of MilliQwater at 400 rpm (until it became a visibly clear solution).

The pharmaceutically active agent solution was combined with thefunctionalized complexation polymer solution at specific ratios as setforth below and monitored for precipitation. The combined materials werestirred for 30 min and then centrifuged in 50 mL Falcon™ tubes. Thesupernatant solution was removed for HPLC analysis to determine thequantity of free pharmaceutically active agent (not complexed). Whereobtained, the resultant precipitate was resuspended in 50-100 mL of 50mM NH₄HCO₃ solution and either lyophilized or spray dried as set forthbelow.

Lyophilization: Aliquots of 3 mL each of the bulk suspension obtainedfrom the above step were transferred into 5 mL, type-BD Hypak™ glasssyringes and lyophilized using the lyophilization cycle of a program P90(optimized for each pharmaceutically active agent) to fit the stepsprovided with FTS lyophilizer (Dura Stop model), MP Stoppering TrayDryer, Stone Ridge, N.Y. The lyophilization cycle is provided in Table 1below. The final amount of complexed pharmaceutically active agent ineach syringe is based on the appropriate dose of the pharmaceuticallyactive agent. The syringes were seal pouched and stored in a −20° C.freezer until further study.

TABLE 1 Lyophilization Cycle SHELF Chamber Modified TEMP TIME pressurelyophilization STEP (° C.) (HOUR) (mT) protocol FREEZING PRECOOL @ −40Not 1 hr prior to load- controlled ing instrument was pre-cooled −40 2.03000 Modified to fit the freezing steps available for FTS LyophilizerPRIMARY −25 2.0 100 DRYING −30 35.0 SECONDARY 25 2.0 Actual time wasDRYING about 25 hours 25 10.0 5 10.0 200 Actual hold time was 8 hours

Spray dry conditions:

-   -   Inlet temperature set up: 140° C.    -   Actual outlet temperature: 50-80° C.    -   Aspirator 100%    -   Pump: 13%    -   Nozzle Cleaner: 3-5 pulses

The active content in the complexed powder was determined by running iton HPLC. The powder was dissolved in 2% phosphoric acid, and the clearsolution was run on HPLC system.

Results

The results of the above cornplexation experiments are provided in partin Table 2 below. The cornplexation efficiency for exenatide and GLP-1analog with an amine functionalized 50:50 copolymer (Example 2H) as afunction of polymer to peptide ratio is provided in FIG. 1.

TABLE 2 Pharmaceutically active agent Complex Ratio Outcome ExenatideAmine 47 mg of No functionalized Peptide, Precipitation homopolymer 30mg of PCL- (Example 2G) NH₂ GLP-1 analog Amine 11.4 mg of Instantaneousfunctionalized Peptide, Precipitation homopolymer 2.7 mg of (>99%)(Example 2G) PCL-NH₂ Exenatide Amine 47 mg of Instantaneousfunctionalized Peptide, Precipitation copolymer, 50:50 30 mg of PCL-(~76%) (Example 2H) NH₂ GLP-1 analog Amine 11.4 mg of Instantaneousfunctionalized Peptide, Precipitation copolymer, 50:50 2.7 mg of (>99%)(Example 2H) PCL-NH₂ Exenatide Carboxylate 10 mg of No functionalizedPeptide, Precipitation homopolymer 20 mg of PCL- (Example 2I) COOH GLP-1analog Carboxylate 10 mg of No functionalized Peptide, Precipitationhomopolymer 20 mg of PCL- (Example 2I) COOH Exenatide Guanidinium 10 mgof No functionalized Peptide Precipitation homopolymer 20 mg of PCL-(Example 2J) GuHCl GLP-1 analog Guanidinium 10 mg of No functionalizedPeptide Precipitation homopolymer 20 mg of PCL- (Example 2J) GuHCl

Example 4: Complexation of Liraglutide with Functionalized ComplexationPolymers

Complexes between liraglutide and functionalized complexation polymerswere prepared as set forth below.

Preparation of Lyophilized Powder of Complexed Liraglutide: 10 mg ofLiraglutide was placed in a 20 mL wide-mouth glass jar. 1 mL of 50 mMNaHCO₃ (pH 9.5) solution was added, and the mixture was stirred at 400rpm for 5 min at room temperature (until it became a visibly clearsolution).

10 mg of Amine-functionalized complexation polymer ((Example 2C,prepared using 5.4 kDa precursor polymer) or (Example 2C, prepared using11.4 kDa precursor polymer) or (Example 2C, prepared using 16.8 kDaprecursor polymer) or (Example 2H, prepared using 17.2 kDa precursorpolymer)) was then added to 1 mL of deionized water, and the mixture wasstirred at 400 rpm (until it became a visibly clear solution). Theweight ratio of peptide to polymer was 1:1.0. 1 mL of 10 mg/mL of aminefunctionalized polymer solution was added to liraglutide solution (ratioof 1:1), and the mixture was stirred at 400 rpm (until it formed a whiteprecipitate). The suspension was stirred for 30 min before centrifugingthe whole suspension in a 50 mL Falcon™ tube and removing thesupernatant solution for HPLC analysis to determine the amount of free(not complexed) active. The resultant precipitate was resuspended in50-100 mL of 50 mM NH₄HCO₃ solution and was lyophilized using theprocedure in Example 3 above.

Example 5: Complexation of Liraglutide with Functionalized ComplexationPolymer (AFCP-1)

Complexes between liraglutide and functionalized complexation polymerswere prepared as set forth below.

Preparation of Lyophilized powder of Complexed Liraglutide: 92.3 mg ofliraglutide were placed in a 20 mL wide-mouth glass jar. 6.5 mL of 50 mMNaHCO₃ (pH 9.5) solution were added, and the mixture was stirred at 400rpm for 5 min at room temperature (until it became a visibly clearsolution).

203.7 mg of Amine-functionalized complexation polymer, AFCP-1 (Example2A, prepared using 15 kDa precursor polymer) were then added to 20 mL ofdeionized water, and the mixture was stirred at 400 rpm (until it becamea visibly clear solution). The weight ratios of peptide to polymer were1:0.3, 1:0.6, 1:0.8, 1:1.0, and 1:1.4, respectively. By way of example,9.2 mL of 10 mg/mL AFCP-1 solution were added to liraglutide solution(ratio of 1:0.8), and the mixture was stirred at 400 rpm (until itformed a white precipitate). The suspension was stirred for 30 minbefore centrifuging the whole suspension in a 50 mL Falcon™ tube andremoving the supernatant solution for HPLC analysis to determine howmuch of active was free (not complexed). The resultant precipitate wasresuspended in 50-100 mL of 50 mM NH₄HCO₃ solution, and the mixture waslyophilized using the procedure in Example 3 above.

The complexation efficiencies for liraglutide with the aminefunctionalized PCL copolymer (AFCP-1) prepared in Example 2A as afunction of polymer to peptide ratio are provided in FIG. 2.

Example 6: Complexation of Liraglutide with Functionalized ComplexationPolymer (AFCP-2)

Complexes between liraglutide and functionalized complexation polymerswere prepared as set forth below.

Preparation of Lyophilized Powder of Complexed Liraglutide: 107.3 mg ofliraglutide were placed in a 60 mL wide-mouth glass bottle. 10.7 mL of50 mM NH₄HCO₃ (pH 8.12) solution were added, and the mixture was stirredat 400 rpm for 30 min at room temperature (until it became a visiblyclear solution).

276.4 mg of Amine-functionalized cornplexation polymer, AFCP-2 (Example2A, 44 kD) were then added to 27.6 mL of deionized water, and themixture was stirred at 400 rpm (until it became a visibly clearsolution). The weight ratios of peptide to polymer were 1:0.3, 1:0.6,1:0.8 and 1:1.0, respectively. By way of example, 10.73 mL of 10 mg/mLAFCP-2 solution were added to liraglutide solution (1:1 ratio), stirredat 400 rpm (until it formed a white precipitate) and kept at −10° C.overnight to complete the precipitation. The whole suspension wascentrifuged in a 50 mL Falcon™ tube, and the supernatant solution wasremoved for HPLC analysis to determine how much of active was free (notcomplexed). The resultant precipitate was resuspended in 50-100 mL of 50mM NH₄HCO₃ solution, and the mixture was vortexed and sonicated for 10min before placing it in a lyophilizer for lyophilization using theprocedure in Example 3 above.

The complexation efficiencies for liraglutide with the aminefunctionalized PCL copolymer AFCP-2 prepared in Example 2A as a functionof polymer to peptide ratio are provided in FIG. 2.

Example 7 (Control) Complexation of Liraglutide with Zn/Protamine

Complexes between liraglutide and Zn/Protamine were prepared as setforth below.

Preparation of Spray Dried Powder of Liraglutide Complexed withZn/Protamine: 104.9 mg of liraglutide were placed in a 60 mL wide-mouthglass jar. 6.5 mL of 50 mM NH₄HCO₃ (pH 8.13) solution were added, andthe mixture was stirred at 400 rpm for 30 min at room temperature (untilit became a visibly clear solution).

100 mM Zinc acetate dehydrate solution was added to 20 mL of deionizedwater. The molar ratio of peptide to Zn was 1:2. 556 μL of 100 mM Zincacetate solution were added to the liraglutide solution, and the mixturewas stirred at 400 rpm (until it formed a white precipitate).Subsequently, 6.2 mL of 10 mg/mL protamine sulfate solution in waterwere added to the Zinc-Lira suspension. The suspension was allowed tostir for 30 min before centrifuging the whole suspension in a 50 mLFalcon tube and removing the supernatant solution for HPLC analysis todetermine how much of active was free (not complexed). The resultantprecipitate was resuspended in 50-100 mL of 50 mM NH₄HCO₃ solution andwas spray-dried.

Spray dry conditions:

-   -   Inlet temperature set up: 140° C.,    -   Actual outlet temperature: 50-80° C.,    -   Aspirator 100%,    -   Pump: 13%,    -   Nozzle Cleaner: 3 to 5 pulses

Example 8: In Vitro Analysis of Exenatide Complexed with anAmine-Functionalized Copolymer

Dissolution experiments were conducted as set forth below to determinethe in vitro release of exenatide from a polymer complex including anamine functionalized 50:50 copolymer (Example 2H).

Materials and Methods

1.00 g of exenatide powder was placed in a 150 mL wide-mouth glass jar.100 mL of 50 mM NH₄HCO₃ solution were added, and the mixture was stirredat 400 rpm for 30 min at room temperature (until it became a visiblyclear solution).

100 mL of a 10 mg/mL solution of functionalized complexation polymerwere then prepared by stirring 1.00 g of polymer in 100 mL of MilliQwater at 400 rpm (until it became a clear solution).

The exenatide solution was combined with the complexation polymersolution at specific ratios as set forth below and monitored forprecipitation. The combined materials were stirred for 30 min and thencentrifuged in 50 mL Falcon™ tubes. The supernatant solution was removedfor HPLC analysis to determine the quantity of free pharmaceuticallyactive agent (not complexed). The resultant precipitate was resuspendedin 50-100 mL of 50 mM NH₄HCO₃ solution and lyophilized according to themethod described in Example 3.

Lyophilized complex powder was suspended in 1 mL ammonium bicarbonate(50 mM).

For dissolution or in vitro release testing, PBS (Phosphate bufferedsaline) pH˜7.4 was used as the medium, and the complexed powder wasdispersed into the dissolution medium. For the dissolution study, aknown amount of complexed powder was placed into 2 mL conical shapedpolypropylene vials. 1 mL release medium (0.01M PBS at pH 7.4equilibrated at 37° C.) was added gently to each vial such that thesurface of the formulation was not disturbed. The samples were placed at37° C./100 rpm in an orbital shaker. At every time point essentially allthe release medium was removed and replaced by fresh solution. Theamount of exenatide in solution at each time point was determined byHPLC.

Results

The in vitro dissolution profile for exenatide from the polymer complexis provided in FIG. 3. The in vitro dissolution rate forexenatide:PεCL-NH₂ was somewhat increased relative to theexenatide:Zn²⁺:protamine complex prepared as described in Example 7.

Example 9: In Vitro Analysis of a GLP-1 Analog Complexed withAmine-Functionalized Copolymers

Dissolution experiments were conducted as set forth below to determinethe in vitro release of a GLP-1 analog from polymer complexes includingan amine-functionalized 50:50 copolymer (Example 2H) and anamine-functionalized homopolymer (Example 2G), respectively.

Materials and Methods

1.00 g of GLP-1 analog powder was placed in a 150 mL wide-mouth glassjar. 100 mL of 50 mM NH₄HCO₃ solution were added, and the mixture wasstirred at 400 rpm for 30 min at room temperature (until it became avisibly clear solution).

100 mL of a 10 mg/mL solution of functionalized complexation polymerwere then prepared by stirring 1.00 g of polymer in 100 mL of MilliQwater at 400 rpm (until it became a clear solution).

The GLP-1 analog solution was combined with the complexation polymersolution at specific ratios as set forth below and monitored forprecipitation. The combined materials were stirred for 30 min and thencentrifuged in 50 mL Falcon™ tubes. The supernatant solution was removedfor HPLC analysis to determine the quantity of free pharmaceuticallyactive agent (not complexed). The resultant precipitate was resuspendedin 50-100 mL of 50 mM NH₄HCO₃ solution and spray dried according to themethod described in Example 3.

Spray dried complex powder was suspended in 1 mL ammonium bicarbonate(50 mM).

For dissolution or in vitro release testing, PBS (Phosphate bufferedsaline) pH˜7.4 was used as the medium, and the complexed powder wasdispersed into the dissolution medium. For the dissolution study, aknown amount of complexed powder was placed into 2 mL conical shapedpolypropylene vials. 1 mL release medium (0.01 M PBS at pH 7.4equilibrated at 37° C.) was added gently to each vial such that thesurface of the formulation was not disturbed. The samples were placed at37° C./100 rpm in an orbital shaker. At every time point essentially allthe release medium was removed and replaced by fresh solution. Theamount of GLP-1 analog in solution at each time point was determined byHPLC.

Results

The in vitro dissolution profile for the GLP-1 analog from the polymercomplexes is provided in FIG. 4. The in vitro dissolution rate for GLP-1analog from the amine-functionalized homopolymer complex was increasedrelative to that for the amine-functionalized copolymer complex.

Example 10 (Control): In Vitro Analysis of Risperidone Mixed with aCarboxylate-Functionalized Copolymer

Dissolution experiments were conducted as set forth below to determinethe in vitro release from a mixture of risperidone and acarboxylate-functionalized 50:50 copolymer prepared as described inExample 2E. Although the risperidone and copolymer mixture wasoriginally believed to involve a complex, further analysis indicatedthat the risperidone and copolymer precipitated separately at therelevant pH. This observation is mainly due to the limited solubilitiesof risperidone and the copolymer in the reaction mixture at pH˜5.

Materials and Methods

1.00 g of a risperidone powder was placed in a 150 mL wide-mouth glassjar. 100 mL of 50 mM NH₄COOCH₃ solution were added, and the mixture wasstirred at 400 rpm for 30 min at room temperature (until it became avisibly clear solution).

100 mL of a 10 mg/mL solution of functionalized polymer were thenprepared by stirring 1.00 g of polymer in 100 mL of MilliQ water at 400rpm (until it became a clear solution).

The pharmaceutically active agent solution was combined with the polymersolution at specific ratios as set forth below and monitored forprecipitation. The combined materials were stirred for 30 min and thencentrifuged in 50 mL Falcon™ tubes. The supernatant solution was removedfor HPLC analysis to determine the quantity of free pharmaceuticallyactive agent (not precipitated). The resultant precipitate wasresuspended in 50-100 mL of 50 mM NH₄COOCH₃ solution and lyophilizedaccording to the method described in Example 3.

Lyophilized powder was suspended in 1 mL ammonium bicarbonate (50 mM).

For dissolution or in vitro release testing, PBS (Phosphate bufferedsaline) pH˜7.4 was used as the medium, and the powder was dispersed intothe dissolution medium. For the dissolution study, a known amount ofpowder was placed into 2 mL conical shaped polypropylene vials. 1 mLrelease medium (0.01M PBS at pH 7.4 equilibrated at 37° C.) was addedgently to each vial such that the surface of the formulation was notdisturbed. The samples were placed at 37° C./100 rpm in an orbitalshaker. At every time point essentially all the release medium wasremoved and replaced by fresh solution. The amount of risperidone insolution at each time point was determined by HPLC.

Results

The in vitro dissolution profile for risperidone from the mixture withthe polymer is provided in FIG. 5. The in vitro dissolution rate wasrelatively low with a cumulative release of between 4% and 8% out to 130hours. Most of the release from risperidone polymer mixture is due toslow dissolution of risperidone at pH 7.4

Example 11: In Vitro Analysis of Liraglutide Complexed withAmine-Functionalized Copolymers

Dissolution experiments were conducted as set forth below to determinethe in vitro release of liraglutide from a polymer complex including anamine-functionalized 50:50 copolymer.

Materials and Methods

1.00 g of liraglutide powder was placed in a 150 mL wide-mouth glassjar. 100 mL of 50 mM NH₄HCO₃ solution were added, and the mixture wasstirred at 400 rpm for 30 min at room temperature (until it became avisibly clear solution).

100 mL of a 10 mg/mL solution of functionalized cornplexation polymerwere then prepared by stirring 1.00 g of polymer in 100 mL of MilliQwater at 400 rpm (until it became a clear solution).

The liraglutide solution was combined with the cornplexation polymersolution at specific ratios as set forth below and monitored forprecipitation. The combined materials were stirred for 30 min and thencentrifuged in 50 mL Falcon™ tubes. The supernatant solution was removedfor HPLC analysis to determine the quantity of free pharmaceuticallyactive agent (not complexed). The resultant precipitate was resuspendedin 50-100 mL of 50 mM NH₄HCO₃ solution and spray dried according to themethod described in Example 3.

Complexes based on four different amine-functionalized 50:50 copolymerswere tested as described below. Three of these copolymers were preparedaccording to Example 2C, the precursor polymers having molecular weightsof 5.4 kDa, 11.4 kDa, and 16.8 kDa, respectively. In addition, a complexbased on the amine-functionalized 50:50 copolymer (Example 2H, preparedusing 16.0 kDa precursor polymer) was tested.

Spray dried complex powder was suspended in 1 mL ammonium bicarbonate(50 mM).

For dissolution or in vitro release testing, PBS (Phosphate bufferedsaline) pH˜7.4 was used as the medium, and the complexed powder wasdispersed into the dissolution medium. For the dissolution study, aknown amount of complexed powder was placed into 2 mL conical shapedpolypropylene vials. 1 mL release medium (0.01M PBS at pH 7.4equilibrated at 37° C.) was added gently to each vial such that thesurface of the formulation was not disturbed. The samples were placed at37° C./100 rpm in an orbital shaker. At every time point essentially allthe release medium was removed and replaced by fresh solution. Theamount of liraglutide in solution at each time point was determined byHPLC.

Results

The in vitro dissolution profiles for liraglutide from the polymercomplexes are provided in FIG. 6. The in vitro dissolution rates forliraglutide from the complexes based on copolymers prepared using the5.4 kDa, 11.4 kDa, and 16.8 kDa precursor polymers were reduced relativeto the dissolution rate for liraglutide from the complex based on theExample 2H copolymer prepared using the 16.0 kDa precursor polymer, butgreater than that for liraglutide from a Zn:Protamine based complexprepared according to Example 7.

Example 12: In Vitro Analysis of Decitabine and AzaCytidine Complexedwith an Amine-Functionalized Copolymer

Dissolution experiments were conducted as set forth below to determinethe in vitro release of decitabine and azacytidine from a polymercomplex including an amine functionalized copolymer (Example 2H).

Materials and Methods

1.00 g of decitabine or azacytidine powder was placed in a 150 mLwide-mouth glass jar. 100 mL of 50 mM NH₄HCO₃ solution were added, andthe mixture was stirred at 400 rpm for 30 min at room temperature (untilit became a visibly clear solution).

100 mL of a 10 mg/mL solution of functionalized complexation polymerwere then prepared by stirring 1.00 g of polymer in 100 mL of MilliQwater at 400 rpm (until it became a clear solution).

The decitabine or azacytidine solution was combined with thecomplexation polymer solution at specific ratios as set forth below andmonitored for precipitation. The combined materials were stirred for 30min and then centrifuged in 50 mL Falcon™ tubes. The supernatantsolution was removed for HPLC analysis to determine the quantity of freepharmaceutically active agent (not complexed). The resultant precipitatewas resuspended in 50-100 mL of 50 mM NH₄HCO₃ solution and lyophilizedaccording to the method described in Example 3.

Lyophilized complex powder was suspended in 1 mL ammonium bicarbonate(50 mM).

For dissolution or in vitro release testing, PBS (Phosphate bufferedsaline) pH˜7.4 was used as the medium, and the complexed powder wasdispersed into the dissolution medium. For the dissolution study, aknown amount of complexed powder was placed into 2 mL conical shapedpolypropylene vials. 1 mL release medium (0.01M PBS at pH 7.4equilibrated at 37° C.) was added gently to each vial such that thesurface of the formulation was not disturbed. The samples were placed at37° C./100 rpm in an orbital shaker. At every time point essentially allthe release medium was removed and replaced by fresh solution. Theamount of decitabine or azacytidine in solution at each time point wasdetermined by HPLC.

Results

The in vitro dissolution profiles for decitabine and azacytidine fromthe polymer complexes are provided in FIG. 7. However, under the testedconditions, decitabine and azacytidine are believed to be significantlydegraded, possibly due to the relative instability of these compounds inwater. Accordingly, the curves in FIG. 7 do not accurately represent thedissolution of intact decitabine and azacytidine from the polymercomplex. Further analysis of decitabine complexation and dissolution wasperformed as described below in Example 13.

Example 13: Complexation of Decitabine with Functionalized Copolymersand Dissolution Analysis in Connection with Same

Complexation of decitabine with various functionalized copolymers wasattempted, and the resulting product was analyzed for in vitrodissolution characteristics as described below.

Materials and Methods

Decitabine powder (5 mg each) was dissolved in 100 μL of DMSO and mixedwith different weight ratios (e.g., 5.0 mg for a 1:1 ratio) ofcomplexation polymer in 1 mL of water for each of AFCP-1, AFCP-2, CFCP-1and CFCP-4. The weight ratios of decitabine to polymer were 1:1, 1:1,1:1 and 1:4, respectively.

After mixing each solution was vortexed for 20s and centrifuged toseparate the precipitate from any un-reacted polymer and decitabine inthe supernatant.

The precipitates were lyophilized to dryness. Known amounts ofdecitabine-polymer complex were dispersed in 1 mL of PBS and placed onan orbital shaker at 37° C./100 rpm. Dissolution of the complex wasmonitored over time, with complete replacement of the aqueous medium atevery time point.

Results

The tested complexation conditions resulted in relatively poordecitabine complex yields. The results of the dissolution assay areprovided in FIGS. 9 and 10 and further demonstrate the unstable natureof decitabine complexed with both amine-functionalized andcarboxyl-functionalized complexable polymers (AFCP and CFCP). While theAFCP-decitabine complexes dissolved faster in PBS at 37° C. than theCFCP-decitabine complex, both complexes were relatively unstable underthe tested aqueous conditions.

Example 14: In Vitro Analysis of GLP-1 Analog Complexed with a Non-ClickChemistry-Based Functionalized Polymer

Dissolution experiments were conducted as set forth below to determinethe in vitro release of a GLP-1 analog from a polymer complex includinga non-click chemistry based hydrazide-functionalized polymer(NH₂—NH—PLGA-NH—NH₂, 5 kDa) available from Polyscitech, West Lafayette,Ind.

Materials and Methods

1.00 g of GLP-1 analog powder was placed in a 150 mL wide-mouth glassjar. 100 mL of 50 mM NH₄HCO₃ solution were added, and the mixture wasstirred at 400 rpm for 30 min at room temperature (until it became avisibly clear solution).

100 mL of a 10 mg/mL solution of functionalized complexation polymerwere then prepared by stirring 1.00 g of polymer in 100 mL of MilliQwater at 400 rpm (until it became a clear solution).

The GLP-1 analog solution was combined with the complexation polymersolution at specific ratios as set forth below and monitored forprecipitation. The combined materials were stirred for 30 min and thencentrifuged in 50 mL Falcon™ tubes. The supernatant solution was removedfor HPLC analysis to determine the quantity of free pharmaceuticallyactive agent (not complexed). The resultant precipitate was resuspendedin 50-100 mL of 50 mM NH₄HCO₃ solution and spray dried according to themethod described in Example 3.

Spray dried complex powder was suspended in 1 mL ammonium bicarbonate(50 mM).

For dissolution or in vitro release testing, PBS (Phosphate bufferedsaline) pH˜7.4 was used as the medium, and the complexed powder wasdispersed into the dissolution medium. For the dissolution study, aknown amount of complexed powder was placed into 2 mL conical shapedpolypropylene vials. 1 mL release medium (0.01M PBS at pH 7.4equilibrated at 37° C.) was added gently to each vial such that thesurface of the formulation was not disturbed. The samples were placed at37° C./100 rpm in an orbital shaker. At every time point essentially allthe release medium was removed and replaced by fresh solution. Theamount of GLP-1 analog in solution at each time point was determined byHPLC.

Results

The in vitro dissolution profiles for the GLP-1 analog from the polymercomplexes are provided in FIG. 8 along with the dissolution profile ofthe GLP-1 analog from a Zn:protamine complex prepared as described inExample 7. In FIG. 8, “SUPE”=supernatant and “PPT”=precipitate, wherePPT represents polymer complexed with the GLP-1 analog and SUPErepresents primarily GLP-1 analog+uncomplexed polymer. The PPT (complex)showed controlled release up to 7 days, while the SUPE showed asignificantly more rapid release.

Example 15: In Vitro Analysis of Liraglutide Complexed with AdditionalAmine-Functionalized Copolymers

Dissolution experiments were conducted as set forth below to determinethe in vitro release of liraglutide from a polymer complex including anamine-functionalized copolymer.

Materials and Methods

1.00 g of liraglutide powder was placed in a 150 mL wide-mouth glassjar. 100 mL of 50 mM NH₄HCO₃ solution were added, and the mixture wasstirred at 400 rpm for 30 min at room temperature (until it became avisibly clear solution).

100 mL of a 10 mg/mL solution of functionalized complexation polymerwere then prepared by stirring 1.00 g of polymer in 100 mL of MilliQwater at 400 rpm (until it became a visibly clear solution). Thefunctionalized complexation polymers were two differentamine-functionalized PCL copolymers, which were prepared according toExample 2A (AFCP-1 and AFCP-2).

The liraglutide solution was combined with the complexation polymersolution at specific ratios as set forth below and monitored forprecipitation. The combined materials were stirred for 30 min and thencentrifuged in 50 mL Falcon™ tubes. The supernatant solution was removedfor HPLC analysis to determine the quantity of free pharmaceuticallyactive agent (not complexed). The resultant precipitate was resuspendedin 50-100 mL of 50 mM NH₄HCO₃ solution and lyophilized according to themethod described in Example 3.

Lyophilized complex powder was suspended in 1 mL ammonium bicarbonate(50 mM).

For dissolution or in vitro release testing, PBS (Phosphate bufferedsaline) pH˜7.4 was used as the medium, and the complexed powder wasdispersed into the dissolution medium. For the dissolution study, aknown amount of complexed powder was placed into 2 mL conical shapedpolypropylene vials. 1 mL release medium (0.01M PBS at pH 7.4equilibrated at 37° C.) was added gently to each vial such that thesurface of the formulation was not disturbed. The samples were placed at37° C./100 rpm in an orbital shaker. At every time point essentially allthe release medium was removed and replaced by fresh solution. Theamount of liraglutide in solution at each time point was determined byHPLC.

Results

The in vitro dissolution profiles for liraglutide from the polymercomplexes are provided in FIGS. 11 and 12 relative to those foruncomplexed liraglutide (Lira) and liraglutide complexed withZn:Protamine prepared in accordance with Example 7.

Example 16: In Vivo Analysis of Liraglutide Complexed withAmine-Functionalized Copolymers

The following materials and methods were utilized in rat PK studies.

Materials and Methods

1.00 g of liraglutide powder was placed in a 150 mL wide-mouth glassjar. 100 mL of 50 mM NH₄HCO₃ solution were added, and the mixture wasstirred at 400 rpm for 30 min at room temperature (until it became avisibly clear solution).

For each of two polymers, 100 mL of a 10 mg/mL solution offunctionalized complexation polymer were then prepared by stirring 1.00g of polymer in 100 mL of MilliQ water at 400 rpm (until it became aclear solution). The functionalized complexation polymers were twodifferent amine-functionalized copolymers, which were prepared accordingto Example 2A (AFCP-1 and AFCP-2).

The liraglutide solution was combined with the complexation polymersolution at specific ratios as set forth below and monitored forprecipitation. The combined materials were stirred for 30 min and thencentrifuged in 50 mL Falcon™ tubes. The supernatant solution was removedfor HPLC analysis to determine the quantity of free pharmaceuticallyactive agent (not complexed). The resultant precipitate was resuspendedin 50-100 mL of 50 mM NH₄HCO₃ solution and lyophilized according to themethod described in Example 3.

Lyophilized complex powder was suspended in 1 mL ammonium bicarbonate(50 mM) or a vehicle comprising benzyl benzoate (BB)/poly (DL-Lactide)(Mw 15,100 Da) (PLA) in a 90/10 weight ratio, which was previouslyprepared by mixing BB and PLA in the desired ratio.

Liraglutide complexed powders were reconstituted in aqueous andnon-aqueous vehicles prior to SC injections in rats. The followingformulations were prepared for use in the in vivo experiments.

-   -   F1. aqueous solution of Liraglutide (Bachem) in 50 mM NH₄HCO₃    -   F2. aqueous slurry of Lira-Zn/Pro(1/2/0.3, m/m) in 50 mM NH₄HCO₃    -   F3. aqueous slurry of Lira-AFCP-1 (1/1 wt/wt) in 50 mM NH₄HCO₃    -   F4. aqueous slurry of Lira-AFCP-2 (1/1 wt/wt) in 50 mM NH₄HCO₃    -   F5. non-aqueous slurry of Lira-AFCP-1 (1/1 wt/wt) in BB/PLA        (90/10)    -   F6. non-aqueous slurry of Lira-AFCP-2 (1/1 wt/wt) in BB/PLA        (90/10)    -   F7. non-aqueous slurry of Lira-Zn/Pro(1/2/0.3, m/m) in BB/PLA        (90/10)

The targeted dosing regimen for the in vivo experiments was as providedbelow in Table 3.

TABLE 3 Dose No. and Dose Regimen Group Sex of Dose Volume and BloodCollection No. Animals Treatment (mg/rat)* (μL/rat)* Route (n = 5 pertimepoint) 1 5M Liraglutide in Buffer 2 100 Once, SC Pre-dose (−24 hr),15, (SQ Bolus) 30 min, 1, 2, 4, 8, 12 hours, 1, 2, 3, 5, and 7 days 2 5MLiraglutide + Zn/ Target = 2 100 Once, SC Pre-dose (−24 hr), 15,Protamine in Buffer 30 min, 1, 2, 4, 8, 12 hours, 1, 2, 3, 5, and 7 days3 5M Liraglutide + AFCP- Target = 2 100 Once, SC Pre-dose (−24 hr), 30min, 1 in Buffer 1, 2, 4, 8, 12 hours, 1, 2, 3, 5, 7, 10, 14 and 29 days4 5M Liraglutide + AFCP- Target = 2 100 Once, SC Pre-dose (−24 hr), 30min, 2 in Buffer 1, 2, 4, 8, 12 hours, 1, 2, 3, 5, 7, 10, 14 and 29 days5 5M Liraglutide + AFCP- Target = 2 100 Once, SC Pre-dose (−24 hr), 30min, 1 + BB/PLA 1, 2, 4, 8, 12 (90/10) hours, 1, 2, 3, 5, 7, 10, 14 and29 days 6 5M Liraglutide + AFCP- Target = 2 100 Once, SC Pre-dose (−24hr), 30 min, 2 + BB/PLA 1, 2, 4, 8, 12 (90/10) hours, 1, 2, 3, 5, 7, 10,14 and 29 days 7 5M Liraglutide + Target = 2 100 Once, SC Pre-dose (−24hr), 30 min, Zn/Protamine + 1, 2, 4, 8, 12 BB/PLA (90/10) hours, 1, 2,3, 5, 7, 10, 14 and 29 days Buffer = 50 mM Ammonium Bicarbonate (pH~8.1) *The target 1 week dose of Liraglutide in human is 14 mg. TheVictoza dose is 0.6-1.8 mg/day in 200-300 μL.

Results

PK profiles for liraglutide complexed with amine-functionalizedcopolymers are provided in FIGS. 13 and 14. Dosing rates (mg/kg) variedacross the test groups and among animals within a given test group.Accordingly, the plasma data provided in FIGS. 13 and 14 has been doserate normalized to allow a fairer comparison of the performance of theformulations.

FIGS. 13 and 14 provide geometric mean data for the aqueous and BB:PLAvehicle suspensions of the liraglutide-counter-ion complexes,respectively. A clear progression emerges in the ability of thecounter-ions to control the dissolution of the complexes in aqueoussuspension: AFCP2>AFCP1>Zn2+/protamine (FIG. 13).

A progression also emerges in the contribution of the depot vehicle (orcomplex+vehicle) to overall control of peptide delivery:AFCP2<AFCP1<Zn2+/protamine (FIG. 14).

These data suggest preliminarily that an aqueous suspension ofLira:AFCP2 would be an adequate formulation for weekly delivery.

FIG. 14 emphasizes that the contribution the BB:la-PLA 90:10 (%, w/w)vehicle makes to the control of liraglutide delivery depends strongly onthe particular complex suspended in the vehicle. The vehicle makes astrong contribution in the case of the Zn²⁺/protamine complex, amoderate contribution for AFCP1, and a small (though important)contribution for AFCP2.

Using iv bolus data, the absolute BA of liraglutide obtained from theseformulations was calculated up to the last time point for whichquantifiable data were available for each animal. These results areprovided in FIG. 15. Except for several animals, BA was generally <12%,which is consistent with results obtained in rats for severalliraglutide formulations that comprised free peptide suspended invehicles comprising sucrose acetate isobutyrate (absolute BA ranged from5-28%).

Example 17: Local Tolerability Following Injection in Rats

Histopathological assessment of skins from the rats injected in Example15 was performed using standard procedures. In general, there wasminimal to mole inflammation present in the deep dermis in all groups atboth the vehicle and active sites. This indicates that there is somenonspecific local multifocal inflammation induced by all of the vehiclesand the test article. There were also low numbers of small granulomaspresent in the deep dermis in a few animals in most groups in both thevehicle and active groups. These are considered to be a resolvingforeign body reaction or granuloma formulation reaction to the test andvehicle articles. This indicates that both the test article and thevarious vehicles alone or with test article will induce a small localforeign body reaction or granuloma formation particularly in Groups 3,5, and 6.

Example 18: Preparation of Amine Functionalized Copolymer (30:70 Poly(α-Cl-ε-caprolactone-co-DL-lactide)) (AFCP-3)

The precursor polymer utilized in this Example was the 31.8 kDa polymerprepared according to the method described in Example 1B above.

A solution of the precursor poly(α-chloro-ε-caprolactone-co-DL-lactide)in DMF (4 mL solvent/g polymer) was evacuated and back-filled withnitrogen five times. Sodium azide (NaN₃, 1.2 equiv.) was added andstirred overnight at room temperature via a magnetic stir bar. Thepresence of a new organic azide moiety was confirmed by FT-IR (at 2107cm⁻¹). The flask was cooled to 0° C. in an ice bath and was evacuatedand back-filled with nitrogen five times. Under a stream of nitrogen,propargyl amine (1.0 equiv.) and sodium ascorbate (0.12 equiv.) wereadded sequentially. The flask was again evacuated and back-filled withnitrogen 5 times, then copper sulfate (0.05 equiv.) was added, followedby one additional evacuate/back-fill cycle. The reaction was stirred at0° C. for 1 hour, then overnight at room temperature under nitrogen.FT-IR confirmed the disappearance of the organic azide peak at ˜2107cm⁻¹, indicating the reaction was complete. DMF was removed undervacuum, and the thick green polymer ball was then directly dissolved in0.01 N HCl and dialyzed using regenerated cellulose tubing withMWCO=3500 against 0.01 N HCl for two days, changing the dialysate 2times. The polymer solution was then freeze-dried until dry.

Analysis of the polymer indicated it was impure, so it was thenredissolved in 0.01 N HCl. A metal scavenger (SiliaMetS® Thiol, 8equiv.) was added to the polymer solution, and the mixture was stirredovernight. The scavenger was filtered out, and the resulting filtratewas purified via ultrafiltration in a stirred cell using 1.5 diavolumesof purified water as the dialysate. The purified polymer solution wasthen transferred to VirTis® jars, shell-frozen, and lyophilized untildry. The resulting amine functionalized copolymer had a M_(w) of 31.8kDa (M_(w) of precursor polymer), a Pendant/Polymer ratio of 1.81×10⁻³mol/g, and a % substitution of -30.

Example 19: Preparation of Amine Functionalized Copolymer (12.5:87.5Poly(α-Cl-ε-caprolactone-co-DL-lactide)) (AFCP-4)

The precursor polymer utilized in this Example was the 5.6 kDa polymerprepared according to the method described in Example 1B above.

To a solution of the precursor poly(α-Cl-ε-caprolactone-co-DL-lactide)in N-methylpyrrolidone (NMP; 5 mL solvent/g polymer) was added sodiumazide (NaN₃, 1.2 equiv.) with vigorous stirring via a magnetic stir bar.The flask was evacuated and back-filled with nitrogen five times and wasstirred overnight at room temperature. The presence of a new organicazide moiety was confirmed by FT-IR (at ˜2100 cm⁻¹). The flask wascooled to 0° C. in an ice bath and was evacuated and back-filled withnitrogen five times. Under a stream of nitrogen, propargyl amine (1.0equiv.), triethylamine (0.1 equiv.), and copper iodide (0.1 equiv.) wereadded sequentially. The flask was again evacuated and back-filled withnitrogen. The exothermic reaction stirred at 0° C. for an hour; the icebath was then removed, and the reaction continued to stir vigorouslyovernight under nitrogen. FT-IR confirmed the disappearance of theorganic azide at ˜2100 cm⁻¹, indicating the reaction was complete. Thesolids were filtered out of the reaction mixture and were washed withample DMF. The filtrate was diluted with 0.01N hydrochloric acid untilthe solution was homogeneous and the pH was 4-5. A metal scavenger(SiliaMetS® Thiol, 8 equiv.) was added to the polymer solution, and themixture was stirred overnight. The scavenger was filtered out, and theresulting filtrate was purified via ultrafiltration in a stirred cellusing 1.5 diavolumes of purified water as the dialysate. The purifiedpolymer solution was then transferred to VirTis® jars, shell-frozen, andlyophilized until dry.

The resulting amine functionalized copolymer had a M_(w) of 5.6 kDa(M_(w) of precursor polymer), a Pendant/Polymer ratio of 7.59×10⁻⁴mol/g, and a % substitution of 12.5. The resulting amine functionalizedcopolymer was marginally soluble in aqueous medium at pH 5.

Example 20: Preparation of Amine Functionalized Copolymer (25:60:15Poly(α-Cl-ε-caprolactone-co-DL-lactide-co-glycolide)) (AFCP-5)

The precursor polymer utilized in this Example was the 17.5 kDa polymerprepared according to the method described in Example 1G above.

To a solution of the precursorpoly(α-Cl-ε-caprolactone-co-DL-lactide-co-glycolide) inN-methylpyrrolidone (NMP; 5 mL solvent/g polymer) was added sodium azide(NaN₃, 1.2 equiv.) with vigorous stirring via a magnetic stir bar. Theflask was evacuated and back-filled with nitrogen five times and wasstirred overnight at room temperature. The presence of a new organicazide moiety was confirmed by FT-IR (at ˜2100 cm⁻¹). The flask wascooled to 0° C. in an ice bath and was evacuated and back-filled withnitrogen five times. Under a stream of nitrogen, propargyl amine (1.0equiv.), triethylamine (0.1 equiv.), and copper iodide (0.1 equiv.) wereadded sequentially. The flask was again evacuated and back-filled withnitrogen. The exothermic reaction stirred at 0° C. for an hour; the icebath was then removed, and the reaction continued to stir vigorouslyovernight under nitrogen. FT-IR confirmed the disappearance of theorganic azide at ˜2100 cm⁻¹, indicating the reaction was complete. Thesolids were filtered out of the reaction mixture and were washed withample DMF. The filtrate was diluted with 0.01N hydrochloric acid untilthe solution was homogeneous and the pH was 4-5. A metal scavenger(SiliaMetS® Thiol, 8 equiv.) was added to the polymer solution, and themixture was stirred overnight. The scavenger was filtered out, and theresulting filtrate was purified via ultrafiltration in a stirred cellusing 1.5 diavolumes of purified water as the dialysate. The purifiedpolymer solution was then transferred to VirTis® jars, shell-frozen, andlyophilized until dry.

The resulting amine functionalized copolymer had a M_(w) of 17.5 kDa(M_(w) of precursor polymer), a Pendant/Polymer ratio of 1.48×10⁻³mol/g, and a % substitution of 25. The resulting amine functionalizedcopolymer was insoluble at any pH in purely aqueous media. It waspurified as a solution in 1:1 Acetone/Water.

Example 21: Preparation of Carboxylate Functionalized Copolymer (CFCP-4)

The precursor polymer utilized in this Example was the 15.7 kDa polymerprepared according to the method described in Example 1E above.

A solution of the precursorpoly(α-chloro-ε-caprolactone-co-ε-caprolactone) in NMP (4 mL solvent/gpolymer) was evacuated and back-filled with nitrogen five times. Sodiumazide (NaN₃, 1.2 equiv.) was added and stirred overnight at roomtemperature via a magnetic stir bar. The presence of a new organic azidemoiety was confirmed by FT-IR (at 2105 cm⁻¹). The flask was cooled to 0°C. in an ice bath and was evacuated and back-filled with nitrogen fivetimes. Under a stream of nitrogen, 5-hexynoic acid (1.2 equiv.) andtriethylamine (1.3 equiv.) were added sequentially. The flask was againevacuated and back-filled with nitrogen 5 times and copper iodide (0.1equiv.) was added, followed by one additional evacuate/back-fill cycle.The reaction was stirred at 0° C. for 2 hours, then overnight at roomtemperature under nitrogen. FT-IR confirmed the disappearance of theorganic azide peak, indicating the reaction was complete. The reactionwas filtered through a 0.45 μm hydrophilic PTFE membrane using apressure filter; the solid bed was rinsed with an equal amount of NMP,and the resulting solution was slowly and carefully dissolved (withexcessive foaming) in a saturated aqueous solution of sodium bicarbonateuntil the resulting pH=8. Activated charcoal (Norit® PK3-5, 4-14 mesh,granular; 1:1 carbon to polymer, w:w, ratio) was added and stirredvigorously for 5 hours as an attempt to decolorize the solution. Themixture was refrigerated overnight; it was then warmed to roomtemperature and filtered through a bed of celite and medium porosityfilter paper. The filtrate was purified via TFF with a 3 kDa MWCOmembrane cassette using deionized water as the dialysate. Afterpurification for two days (3.5 diavolumes), the green color stillremained. The solution was then treated with a metal scavenger(SiliaMetS® Thiol, 10 equiv.), and the color began to fade almostimmediately; the mixture was stirred at room temperature overnight. Thescavenger was filtered out, and the resulting filtrate was purified viatangential flow filtration (TFF) using a 3 kDa MWCO membrane anddeionized water as the dialysate. Purification continued until the pH ofthe permeate solution was neutral; a total of 9 diavolumes of deionizedwater was used. The purified polymer solution was then transferred toVirTis® jars, shell-frozen, and lyophilized until dry.

The resulting carboxylate functionalized copolymer had a M_(w) of 15.7kDa (M_(w) of precursor polymer), a Pendant/Polymer ratio of 2.64×10⁻³mol/g, and a % substitution of 44.

Example 22: Preparation of Carboxylate Functionalized Copolymer (CFCP-5)

The precursor polymer utilized in this Example was the 44.0 kDa polymerprepared according to the method described in Example 1F above.

A solution of the precursorpoly(α-chloro-ε-caprolactone-co-ε-caprolactone) in NMP (4 mL solvent/gpolymer) was evacuated and back-filled with nitrogen five times. Sodiumazide (NaN₃, 1.2 equiv.) was added and stirred overnight at roomtemperature via a magnetic stir bar. The presence of a new organic azidemoiety was confirmed by FT-IR (at 2104 cm⁻¹). The flask was cooled to 0°C. in an ice bath and was evacuated and back-filled with nitrogen fivetimes. Under a stream of nitrogen, 5-hexynoic acid (1.2 equiv.) andtriethylamine (0.1 equiv.) were added sequentially. The flask was againevacuated and back-filled with nitrogen 5 times and copper iodide (0.1equiv.) was added, followed by one additional evacuate/back-fill cycle.The reaction was stirred at 0° C. for 3 hours, then overnight at roomtemperature under nitrogen. FT-IR showed the organic azide peak at ˜2104cm⁻¹ was less intense, but was still present. Triethylamine (1.1 equiv.)was added; the flask was evacuated/back-filled with nitrogen 3 times;and copper iodide (0.01 equiv.) was again added. After stirring forthree additional days, FT-IR confirmed the disappearance of the organicazide peak, indicating the reaction was complete. The reaction wasfiltered through a 0.45 μm hydrophilic PTFE membrane using a pressurefilter; the solid bed was rinsed with an equal amount of NMP, and theresulting solution was slowly and carefully dissolved (with excessivefoaming) in a saturated aqueous solution of sodium bicarbonate until theresulting pH=8. A metal scavenger (SiliaMetS® Thiol, 10 equiv.) wasadded to the polymer solution, and the mixture was stirred overnight.The scavenger was filtered out, and the resulting filtrate was purifiedvia tangential flow filtration (TFF) using a 3 kDa MWCO membrane anddeionized water as the dialysate. Purification continued until the pH ofthe permeate solution was neutral; a total of 15 diavolumes of deionizedwater was used. The purified polymer solution was then transferred toVirTis® jars, shell-frozen, and lyophilized until dry.

The resulting carboxylate functionalized copolymer had a M_(w) of 44.0kDa (M_(w) of precursor polymer), a Pendant/Polymer ratio of 2.7×10⁻³mol/g, and a % substitution of 44.

Example 23: Complexation of Human Growth Hormone (hGH) with AmineFunctionalized Copolymers and Dissolution Analysis of Complexed hGH

HGH was complexed with various amine functionalized copolymers andanalyzed for in vitro dissolution characteristics as described below.

Materials and Methods

Recombinant hGH powder (5 mg each) was dissolved in 1 mL of 50 mMAmmonium Bicarbonate (pH˜8.1) and mixed with different weight ratios(2.5, 5, 10 and 20 mg) (1:0.5, 1:1, 1:2 and 1:4) of complexable polymersincluding AFCP-2, AFCP-3 and AFCP-5 in 1 mL of water.

After mixing, each solution was vortexed for 20 sec and centrifuged toremove supernatant containing unreacted polymer and hGH from theprecipitate.

The precipitate was lyophilized until dry and known amounts of hGHcomplexed with polymer were dispersed in 1 mL of PBS at 37° C./100 rpmin an orbital shaker to assess dissolution. The PBS media wasreplenished at every time point.

Results

The results of the complexation reactions are shown in FIGS. 16 and 17.As shown, AFCP-2 exhibited the highest complexation efficiency withrespect to hGH with over 90% of the hGH precipitated. AFCP-3 and AFCP-5each exhibited a complexation efficiency between 30% and 50%. As shown,AFCP-4 did not form a complex under these conditions. Without intendingto be bound by any particular theory, it is possible that the relativelylow level of amine substation in AFCP-4 was insufficient forcomplexation with hGH. FIG. 17 shows the complexation efficiency ofAFCP-2 with hGH at various w/w ratios. As shown, ratios of 1:1 and 1:2resulted in over 92% of the hGH precipitated.

The results of the in vitro dissolution analysis are provided in FIG.18. As shown, each of the AFCP-2-hGH, AFCP-3-hGH, and AFCP-5-hGHcomplexes provided for lower initial release of hGH (within the first 24hours) relative to native hGH, with the AFCP-2-hGH complex providing asignificantly lower initial release than AFCP-3-hGH or AFCP-5-hGHcomplex.

Example 24: Complexation of Liraglutide with Amine FunctionalizedCopolymers AFCP-2 and AFCP-3

Liraglutide was complexed with AFCP-2 and AFCP-3 amine functionalizedcopolymers at different weight ratios and complexation efficiency wasdetermined.

Materials and Methods

Liraglutide (5 mg) was dissolved in 1 mL of 50 mM NH₄HCO₃ (pH 8.1) andmixed with AFCP-2 or AFCP-3 in 1 mL of water, at weight ratios 1:0.25,1:0.5, 1:1 and 1:2. AFCP-2 is 45% functionalized, M_(w)˜44 kD PCL;AFCP-3 is 30% functionalized, 30:70 PCLL, M_(w)˜32 kD.

After mixing, each preparation was vortexed for 20s and centrifuged toseparate supernatant containing un-reacted peptide and polymer from theprecipitate. Supernatant samples were diluted 10× in water to quantifyun-reacted peptide via RP-HPLC.

Results

The results of the complexation reactions are shown in FIGS. 19 and 20.FIG. 19 shows the % Liraglutide precipitated using AFCP-2 and AFCP-3 atthe various weight ratios. As shown, the % Liraglutide precipitatedusing AFCP-2 was relatively constant across the different weight ratios,while the complexation efficiency of AFCP-3 increased with an increasein the relative amount of the AFCP-3 polymer. FIG. 20 shows the fractionprecipitated based on the molar ratio Liraglutide to AFCP-2 and AFCP-3.

Example 25: Complexation of Immunoglobulin G (IgG) with AmineFunctionalized Copolymers and Dissolution Analysis of Complexed IgG

IgG was complexed with various amine functionalized copolymers andanalyzed for in vitro dissolution characteristics as described below.

Materials and Methods

IgG powder (5 mg each) was dissolved in 1 mL of 50 mM NH₄HCO₃ (pH 8.1)and mixed with a different amine functionalized PCL polymer (AFCP-2,AFCP-3, AFCP-4 and AFCP-5) at 5 mg in 1 mL of water.

After mixing, each solution was vortexed for 20 sec and centrifuged toremove supernatant containing unreacted polymer and IgG from theprecipitate.

The precipitate was lyophilized until dry and known amounts ofIgG-polymer complex were dispersed in 1 mL of PBS at 37° C./100 rpm inan orbital shaker to assess dissolution. The PBS media was replenishedat every time point.

Results

The results of the dissolution assays are shown in FIG. 21. Initialrelease of IgG (within the first 24 hours) was lowest for AFCP-3.Initial release of IgG for AFCP-2 and AFCP-4 complexes was higher thanfor AFCP-3, but lower than for AFCP-5, which showed a relatively highinitial release of IgG (about 80% cumulative release within the first 24hours).

Example 26: Complexation of a Somatostatin Analogue with a CarboxylateFunctionalized Copolymer and Dissolution Analysis of ComplexedSomatostatin Analogue

A synthetic somatostatin analogue was complexed with a carboxylatefunctionalized copolymer (CFCP-1) and analyzed for in vitro dissolutioncharacteristics as described below.

Materials and Methods

Somatostatin analogue powder (5 mg) was dissolved in 1 mL of NH₄HCO₃ andmixed 1:1 (w/w) with COOH-functionalized CFCP-1(50% pendant, Mw˜15.9 kDaPCL (M_(w) of precursor polymer)) in 1 mL of water.

After mixing, each preparation was vortexed for 20 sec and centrifugedto remove supernatant containing unreacted polymer and somatostatinanalogue from the precipitate.

The precipitate was lyophilized until dry and a known amount ofsomatostatin analogue-CFCP-1 complex was suspended in 1 mL of PBS at 37°C./100 rpm in an orbital shaker to assess dissolution. The PBS media wasreplenished at every time point.

Results

The results of the dissolution assay are shown in FIG. 22, whichcompares the % cumulative release of the somatostatin analogue from thesomatostatin analogue-CFCP-1 complex relative to the somatostatinanalogue alone in powder form. As seen in FIG. 22, the somatostatinanalogue-CFCP-complex powder dissolves in PBS at a slower rate than thesomatostatin analogue alone in powder form, making the complex a viableoption for an extended release depot, e.g., for the delivery of asomatostatin analogue over a one month period.

Example 27: Complexation of Cromolyn with a Amine FunctionalizedCopolymers and Dissolution Analysis of Complexed Cromolyn

Cromolyn was complexed with various amine functionalized copolymers andanalyzed for in vitro dissolution characteristics as described below.

Materials and Methods

Cromolyn powder (5 mg each) was dissolved in 1 mL of Ammonium Acetateand mixed with 5.0 mg (1:1) of complexable polymer (AFCP-2, AFCP-3,AFCP-4 and AFCP-5) in 1 mL of water.

After mixing, each preparation was vortexed for 20s, then centrifuged toseparate supernatant, containing un-reacted cromolyn and polymer, fromthe precipitate.

Recovered precipitate was then lyophilized to dryness, and itsdissolution rate in aqueous medium assessed: A known amount ofCromolyn-AFCP complex was suspended in 1 mL of PBS at 37° C. on anorbital shaker. The % cumulative release of cromolyn over time wasfollowed, with complete medium replacement at every time point.

Results

The results of the dissolution assays are shown in FIG. 23. Cromolyncomplexed with AFCP2 dissolves in PBS at 37° C. at a slower rate thancromolyn complexed with the other AFCP complexes (30% initial releasecompared to 50-80%), which makes it a viable option for a depotadministered on a weekly basis.

Example 28: Solubility of AFCP-2 and CFCP-1

Solubility of AFCP-2 and CFCP-1 copolymers in water at various pH valueswas determined.

Materials and Methods

˜40 mg/mL of AFCP-2 or CFCP-1 in water was provided at room temperatureand sonicated for 10 min. To the above solutions, either 0.05 N sodiumHydroxide solution or 0.05N Hydrochloric acid solution was added toincrease or decrease the pH of the starting solutions as measured byOrion 3 Star pH Benchtop (Thermo Electron Corporation). The physicalappearance was observed visually.

Results

The results of the above measurements are provided in Table 4 below.

TABLE 4 AFCP-2 (40 mg/mL) CFCP-1 (40 mg/mL) pH Physical Appearance pHPhysical Appearance 2.6 Solution 1.3 Colloidal 2.9 Solution 2.6Colloidal 3.0 Solution 3.3 Colloidal 3.4 Solution 5.9 Colloidal 7.0Solution 6.1 Colloidal 9.4 Solution 6.7 Solution 11.9 Solution 8.2Solution Solution 9.1 Solution

Example 29: Computational Toxicology Assessment

Degradation products for select amine and carboxylate functionalizedpolymers containing a 1,2,3 triazole were analyzed using Derek Nexussoftware in the Lhasa Knowledge Suite available from Lhasa Limited,Leeds, U.K. The Derek Nexus prediction includes an overall conclusionabout the likelihood of toxicity in a structure and detailed reasoninginformation for the likelihood. The prediction is generated by applyingexpert knowledge rules in toxicology to the data returned from thecertified Lhasa knowledge base.

The above structures were analyzed using the Derek Nexus software, andno alerts for mutagenicity, genotoxicity, chromosomal damage orcarcinogenicity were present in connection with the_1,2,3 triazoledegradation products.

Example 30: Stability of AFCP and CFCP-complexed Active Agents FollowingExposure to Gamma Radiation

Glatiramer bromide, insulin, liraglutide, and human growth hormone (hGH)were complexed with AFCP-2, CFCP-5, AFCP-2, and AFCP-2 respectively andanalyzed for stability following exposure to gamma radiation.

Materials and Methods

Active pharmaceutical ingredients (APIs) including Liraglutide(Bachem, >99% pure), Human Growth Hormone (HGH, Biosolutions, >95%pure), Poly (AGLT) Bromide (Sigma, >99% pure) and Insulin (Sigma, >95%pure) were obtained from their respective vendors and dissolved ammoniumbicarbonate buffer (pH ˜8) in appropriate concentrations.Amine-functionalized complexable polymer (AFCP-2) andcarboxyl-functionalized complexable polymer (CFCP-5) were synthesizedand dissolved in MilliQ water in appropriate concentrations (10-20mg/mL). The APIs were mixed with the complexable polymers at a ratio of1:1, 1:4 or 1:10. The instantaneous precipitation was separated fromreaction media (ammonium bicarbonate in water) and dried vialyophilization. The dried powder or the dried powder suspended in BenzylBenzoate were loaded either in glass vial or plastic syringes(Intervene) and packed in aluminum pouches. These samples were exposedto gamma radiation (12.5 kGy-17.5 kGy) for more than 8 hrs at roomtemperature with radiation indicator sticker (Sterigenics, Corona,Calif.). These gamma irradiated samples were then analyzed for purityusing reverse phase liquid chromatography (RPLC) in reference tonon-gamma irradiated samples.

Results

The results of the stability analyses are provided in FIGS. 24-27. Theabove active agents each showed good stability following exposure to 15kGy gamma radiation as evidenced by the relative lack of degradantsshown in the RPLC spectra. As seen in the Figures, non-complexed APIssuch as Liraglutide, HGH, Poly(AGLT) bromide (Glatiramer bromide) andInsulin degraded faster (with a corresponding reduction in potency)after 15 kGy gamma irradiation. These APIs complexed either with AFCP orCFCP showed Gamma irradiation stability (15 kGy) as seen in FIGS. 24-27.RPLC spectra for the uncomplexed, non-irradiated APIs are provided inFIGS. 28 and 29.

The foregoing embodiments and advantages are merely exemplary and arenot to be construed as limiting the present invention. The descriptionof the present invention is intended to be illustrative, and not tolimit the scope of the claims. Many alternatives, modifications, andvariations will be apparent to those skilled in the art.

Having now fully described this invention, it will be understood tothose of ordinary skill in the art that the methods of the presentinvention can be carried out with a wide and equivalent range ofconditions, formulations, and other parameters without departing fromthe scope of the invention or any embodiments thereof.

Aspects, including embodiments, of the present subject matter describedherein may be beneficial alone or in combination, with one or more otheraspects or embodiments. Without limiting the foregoing or subsequentdescription, certain non-limiting aspects of the disclosure numbered1-123 are provided below. As will be apparent to those of skill in theart upon reading this disclosure, each of the individually numberedaspects may be used or combined with any of the preceding or followingindividually numbered aspects. This is intended to provide support forall such combinations of aspects and is not limited to combinations ofaspects explicitly provided below.

ASPECTS OF THE INVENTION

-   1. A complex comprising:    -   a pharmaceutically active agent, and    -   a functionalized polymer, the functionalized polymer comprising        repeat units, the repeat units comprising ionizable repeat units        comprising at least one ionizable side group, a plurality of the        at least one ionizable side groups forming a plurality of        non-covalent bonds with the pharmaceutically active agent,    -   wherein at least 10% of the repeat units comprise at least one        ionizable side group,    -   wherein the functionalized polymer is optionally synthetic,    -   wherein the functionalized polymer is optionally a polyester,    -   wherein the functionalized polymer is optionally linear, and    -   wherein the functionalized polymer optionally has a weight        average molecular weight greater than 15,000 Daltons, as        measured by gel permeation chromatography.-   2. The complex of 1, wherein the at least one ionizable side group    comprises at least one member selected from ammonium, carboxylate,    hydrazinium, guanidinium, sulfate, sulfonate, phosphonate, and    phosphate.-   3. A complex comprising:    -   a pharmaceutically active agent; and    -   a functionalized polymer, the functionalized polymer comprising        repeat units, the functionalized polymer comprising at least one        of: (a) ionizable repeat units comprising at least one ionizable        side group, wherein the at least one ionizable side group        comprises at least one member selected from ammonium,        carboxylate, hydrazinium, guanidinium, sulfate, sulfonate,        phosphonate, and phosphate; and (b) at least one ionizable end        group;    -   wherein a plurality of the at least one ionizable groups form a        plurality of non-covalent bonds with the pharmaceutically active        agent,    -   wherein the functionalized polymer is optionally synthetic,    -   wherein the functionalized polymer is optionally a polyester,    -   wherein the functionalized polymer is optionally linear, and    -   wherein the functionalized polymer optionally has a weight        average molecular weight greater than 15,000 Daltons, as        measured by gel permeation chromatography.-   4. The complex of 3, wherein at least 10% of the repeat units    comprise at least one ionizable side group.-   5. The complex of any one of 1-4, wherein at least 20% of the repeat    units comprise at least one ionizable side group.-   6. The complex of any one of 1-5, wherein at least 40% of the repeat    units comprise at least one ionizable side group.-   7. The complex of any one of 1-6, wherein less than 100% of the    repeat units comprise at least one ionizable side group.-   8. The complex of any one of 1-4, wherein the percentage of the    repeat units comprising at least one ionizable side group ranges    from 10% to 90%.-   9. The complex of any one of 1-4, wherein the percentage of the    repeat units comprising at least one ionizable side group ranges    from 20% to 80%.-   10. The complex of any one of 1-9, wherein the at least one    ionizable side group is covalently bound to the polymer through    click chemistry.-   11. The complex of any one of 1-10, wherein the at least one    ionizable side group is covalently bound to the polymer through    click chemistry catalyzed with copper.-   12. The complex of any one of 1-11, wherein the non-covalent bonds    comprise electrostatic interactions.-   13. The complex of any one of 1-11, wherein the non-covalent bonds    comprise steric interactions.-   14. The complex of any one of 1-11, wherein the non-covalent bonds    comprise hydrogen bonding.-   15. The complex of any one of 1-11, wherein the non-covalent bonds    comprise van der Waals forces.-   16. The complex of any one of 1-15, wherein the complex is a salt.-   17. The complex of any one of 1-16, wherein the pharmaceutically    active agent comprises at least one member selected from a peptide,    protein, and small molecule, the small molecule having a molecular    weight less than 500 Daltons.-   18. The complex of any one of 1-17, wherein the at least one    ionizable side group comprises a positively ionizable side group.-   19. The complex of any one of 1-17, wherein the functionalized    polymer has a net positive charge.-   20. The complex of any one of 1-17, wherein the at least one    ionizable side group comprises a negatively ionizable side group.-   21. The complex of any one of 1-17, wherein the functionalized    polymer has a net negative charge.-   22. The complex of any one of 1-21, wherein the repeat units    comprise repeat units comprising at least one pendant hydrophilicity    modifier.-   23. The complex of 22, wherein the at least one pendant    hydrophilicity modifier is selected from polyethyleneglycol (PEG),    hydroxyl and hydroxyalkyl.-   24. The complex of any one of 1-23, wherein the functionalized    polymer has a weight average molecular weight ranging from 1000    Daltons to 200,000 Daltons, as measured by gel permeation    chromatography.-   25. The complex of any one of 1-24, wherein the functionalized    polymer has a weight average molecular weight ranging from 2000    Daltons to 50,000 Daltons, as measured by gel permeation    chromatography.-   26. The complex of any one of 1-25, wherein the functionalized    polymer has a number average molecular weight ranging from 5000    Daltons to 45,000 Daltons, as measured by NMR spectroscopy.-   27. The complex of any one of 1-26, wherein the functionalized    polymer has a solubility in water of at least 0.01 mg/ml and less    than or equal to 10 mg/ml at 25° C. and pH 7.4.-   28. The complex of any one of 1-27, wherein the functionalized    polymer is biodegradable.-   29. The complex of any one of 1-28, wherein the complex has a    solubility of less than 0.01 mg/mL in water at 25° C. at pH 7.4.-   30. The complex of any one of 1-29, wherein the ratio of the amount    of the pharmaceutically active agent to the amount of the    functionalized polymer in the complex is from 1:1 to 1:10 by weight.-   31. The complex of any one of 1-30, wherein the functionalized    polymer comprises at least one ionizable end group comprising at    least one member selected from ammonium, carboxylate, hydrazinium,    guanidinium, sulfate, sulfonate, and phosphate.-   32. The complex of any one of 1-31, wherein the ionizable repeat    units comprise one or more ionizable side groups that comprise an    optionally substituted heteroarylene ring.-   33. The complex of 32, wherein the optionally substituted    heteroarylene ring is a 1,2,3-triazole ring.-   34. The complex of any one of 1-33, wherein the functionalized    polymer is a polyester.-   35. The complex any one of 1-34, wherein the repeat units comprise    repeat units of the formula (I):

wherein

-   -   m is an integer from 1 to 10, and    -   each R¹ and R² is independently selected from hydrogen,        hydroxyl, optionally substituted alkyl, optionally substituted        alkenyl, optionally substituted alkynyl, optionally substituted        alkoxy, optionally substituted carbocyclyl, optionally        substituted aryl, optionally substituted heterocyclyl,        optionally substituted heteroaryl, and a ionizable side group.

-   36. The complex of 35, wherein the repeat units comprise repeat    units of the formula (I) wherein each R¹ and R² is independently    selected from hydrogen, C₁₋₅ alkyl and a ionizable side group.

-   37. The complex of 35 or 36, wherein the repeat units comprise    repeat units of the formula (I) wherein m is an integer from 1 to 5.

-   38. The complex of any one of 35-37, wherein the repeat units    comprise repeat units of the formula (I) wherein one of the R¹s and    R²s is a ionizable side group and all of the remaining R¹s and R²s    are not ionizable side groups.

-   39. The complex of any one of 35-38, wherein the repeat units    comprise repeat units of the formula (I) wherein m is 5, one R¹ is a    ionizable side group and all of the remaining R¹s and R²s are    hydrogen.

-   40. The complex of any one of 35-39, wherein the ionizable side    group comprises a positively charged side group.

-   41. The complex of any one of 35-39, wherein the ionizable side    group comprises a negatively charged side group.

-   42. The complex of any one of 35-39, wherein the ionizable side    group comprises ammonium, carboxylate, guanidinium, sulfate, or    phosphate.

-   43. The complex of any one of 35-42, wherein the ionizable side    group comprises an optionally substituted heteroarylene ring.

-   44. The complex of any one of 35-43, wherein the ionizable side    group has the formula (II):

wherein R³ comprises a ionizable functional group.

-   45. The complex of 44, wherein R³ comprises at least one member    selected from ammonium, carboxylate, guanidinium, sulfate and    phosphate.-   46. The complex of any one of 35-45, wherein the repeat units    comprise repeat units of the formula (I) wherein m is 5 and all of    the R¹ and R²s are hydrogen.-   47. The complex of any one of 35-46, wherein the repeat units    comprise repeat units of the formula (I) wherein m is 1, R¹ is    hydrogen and R² is hydrogen.-   48. The complex of any one of 35-47, wherein the repeat units    comprise repeat units of the formula (I) wherein m is 1, R¹ is    methyl and R² is hydrogen.-   49. The complex of any one of 35-48, wherein the repeat units    comprise repeat units of the formula (I) wherein m is 2, the R¹ and    R² alpha to the carbonyl group in formula (I) are each hydrogen, the    R¹ beta to the carbonyl group is methyl and the R² beta to the    carbonyl group is hydrogen.-   50. The complex of any one of 35-49, wherein the repeat units    comprise repeat units of the formula (I) wherein m is 3 and all of    the R¹ and R²s are hydrogen.-   51. The complex of any one of 35-50, wherein the repeat units    comprise repeat units of the formula (I) wherein m is 4, and all of    the R¹ and R²s are hydrogen.-   52. The complex of any one of 35-51, wherein the repeat units    comprise repeat units of the formula (I) wherein m is 3, the R¹ and    R² alpha to the carbonyl group in formula (I) are each hydrogen, the    R¹ and R² beta to the carbonyl group are each hydrogen, and the R¹    gamma to the carbonyl group is methyl and the R² gamma to the    carbonyl group is hydrogen.-   53. The complex of any one of 35-52, wherein the functionalized    polymer is a homopolymer of repeat units of formula (I).-   54. The complex of any one of 35-52, wherein the functionalized    polymer is a copolymer comprising at least two different repeat    units.-   55. The complex of 54, wherein each of said at least two different    repeat units is of formula (I).-   56. A composition comprising:    -   a complex comprising:        -   a pharmaceutically active agent, and        -   a functionalized polymer complexed with the pharmaceutically            active agent through non-covalent bonding; and    -   a vehicle,    -   wherein the functionalized polymer is optionally synthetic,    -   wherein the functionalized polymer is optionally a polyester,    -   wherein the functionalized polymer is optionally linear, and    -   wherein the functionalized polymer optionally has a weight        average molecular weight ranging from 2000 Daltons to 20,000        Daltons, as measured by gel permeation chromatography.-   57. The composition of 56, wherein the complex is a complex of any    one of 1-55.-   58. The composition of 56 or 57, wherein the amount of the    functionalized polymer present in the composition is less than 50%    by weight based on the total weight of the composition.-   59. The composition of any one of 56-58, wherein the amount of the    functionalized polymer present in the composition is less than 40%    by weight based on the total weight of the composition.-   60. The composition of any one of 56-59, wherein the amount of the    functionalized polymer present in the composition is less than 30%    by weight based on the total weight of the composition.-   61. The composition of any one of 56-60, wherein the amount of the    functionalized polymer present in the composition is less than 20%    by weight based on the total weight of the composition.-   62. The composition of any one of 56-61, wherein the amount of the    functionalized polymer present in the composition is less than 10%    by weight based on the total weight of the composition.-   63. The composition of any one of 56-62, wherein the amount of the    functionalized polymer present in the composition is less than 5% by    weight based on the total weight of the composition.-   64. The composition of 56 or 57, wherein the amount of the    functionalized polymer present in the composition ranges from 1% by    weight to 50% by weight based on the total weight of the    composition.-   65. The composition of 56 or 57, wherein the amount of the    functionalized polymer present in the composition ranges from 5% by    weight to 50% by weight based on the total weight of the    composition.-   66. The composition of 56 or 57, wherein the amount of the    pharmaceutically active agent present in the composition ranges from    1% to 30% by weight based on the total weight of the composition.-   67. The composition of any one of 56-66, wherein the vehicle    comprises a vehicle polymer that is different from the    functionalized polymer.-   68. The composition of 67, wherein the vehicle polymer is present in    the vehicle in an amount from about 5% to about 40% by weight of the    vehicle.-   69. The composition of any one of 56-68 or 120, wherein the vehicle    comprises a solvent.-   70. The composition of 69, wherein the solvent is present in an    amount ranging from 60% to 100% by weight of the vehicle.-   71. The composition of 69 or 70, wherein the solvent is a    hydrophobic solvent.-   72. The composition of 69 or 70, wherein the solvent is a    hydrophilic solvent.-   73. The composition of 69 or 70, wherein the solvent comprises at    least one member selected from water, buffered aqueous system,    dimethylsulfoxide (DMSO), benzyl alcohol (BA), benzyl benzoate (BB),    hydrogenated castor oil, polyethoxylated castor oil,    dimethylacetamide, ethanol, ethyl acetate, glycofurol, isopropyl    myristate, ethyl benzoate, caprylic/capric triglyceride,    n-methyl-pyrrolidone, propylene glycol monocaprylate, propylene    carbonate, diethyl carbonate, 2-pyrrolidone, triacetin, and triethyl    citrate.-   74. The composition of 69 or 70, wherein the solvent comprises    water.-   75. The composition of any one of 56-68, wherein the vehicle    comprises a buffered aqueous system.-   76. The composition of 75, wherein the buffered aqueous system    comprises at least one of phosphate buffered saline (PBS), ammonium    bicarbonate, cresols, HPMC, ammonium acetate, sulfuric acid, and    HCl.-   77. The composition of any one of 56-76, comprising at least one    member selected from sucrose acetate isobutyrate (SAIB), sucrose,    mannitol, trehalose, surfactant, and antioxidant.-   78. The composition of any one of 56-77, wherein the composition is    free of other complexing agents.-   79. The composition of any one of 56-78, wherein the composition is    free of protamine and, optionally, wherein the composition is free    of polymer other than the functionalized polymer.-   80. The composition of any one of 56-79, wherein the composition is    free of divalent metal ions.-   81. The composition of any one of 56-80, wherein the composition is    free of zinc.-   82. The composition of any one of 56-81, wherein the composition is    free of carboxymethylcellulose (CMC).-   83. A method comprising:    -   providing a precursor polymer comprising repeat units, the        repeat units comprising functionalizable repeat units comprising        at least one functionalizable side group;    -   obtaining a functionalized polymer by transforming, using click        chemistry, said functionalizable repeat units into ionizable        repeat units comprising at least one ionizable side group; and    -   combining the functionalized polymer with a pharmaceutically        active agent to form a complex in which a plurality of the at        least one ionizable side groups form a plurality of non-covalent        bonds with the pharmaceutically active agent.-   84. The method of 83, 121 or 122, wherein the complex is a complex    of any one of 1-55.-   85. The method of 83 or 84, wherein said transforming, using click    chemistry, comprises effecting a cycloaddition reaction.-   86. The method of 85 wherein said cycloaddition reaction is a    Diels-Alder cycloaddition reaction.-   87. The method of 85, wherein said cycloaddition reaction is a    Huisgen 1,3-dipolar cycloaddition reaction.-   88. The method of 85, wherein said cycloaddition reaction is a    cycloaddition reaction between an azide and an alkyne to form a    linkage comprising a 1,2,3-triazole.-   89. The method of any one of 83 to 88, wherein the functionalizable    side group is an azido group and wherein the transforming, using    click chemistry, comprises reacting the precursor polymer with an    alkyne to form the functionalized polymer, the functionalized    polymer comprising at least one 1,2,3-triazole ring.-   90. The method of any one of 83 to 88, wherein the functionalizable    side group is a leaving group and wherein the transforming, using    click chemistry, comprises: (a) transforming the leaving group into    an azido group and thereby providing a polymer intermediate; and (b)    reacting the polymer intermediate with an alkyne to form the    functionalized polymer, the functionalized polymer comprising at    least one 1,2,3-triazole ring.-   91. The method of 90, wherein the leaving group is a halogen group.-   92. The method of 90 or 91, wherein the leaving group is transformed    into an azido group by reacting the precursor polymer with sodium    azide.-   93. The method of any one of 89-92, wherein the alkyne is a terminal    alkyne.-   94. The method of any one of 83 to 88, wherein the functionalizable    side group is an alkynyl group and wherein the transforming, using    click chemistry, comprises reacting the precursor polymer with an    azide to form the functionalized polymer, the functionalized polymer    comprising at least one 1,2,3-triazole ring.-   95. The method of 94, wherein the alkynyl group is a terminal    alkynyl group.-   96. The method of any one of 83-95, wherein the transforming, using    click chemistry, comprises a monovalent copper catalyzed reaction or    a ruthenium catalyzed reaction.-   97. The method of 96, wherein the transforming, using click    chemistry, comprises a monovalent copper catalyzed reaction, and    wherein a monovalent copper catalyst is provided in the reaction    through the ionization of copper iodide or copper bromide.-   98. The method of any one of 83-97, wherein the transforming, using    click chemistry, comprises a copper catalyzed azide-alkyne    cycloaddition reaction.-   99. The method of any one of 83-98, wherein the transforming, using    click chemistry, comprises occurs at least partially under degassing    conditions.-   100. The method of any one of 83-99, wherein the combining occurs at    a temperature ranging from 10° C. to 40° C.-   101. The method of any one of 83-99, wherein the precursor polymer    comprises repeat units of the formula (I′):

wherein

-   -   m′ is an integer from 1 to 10, and    -   each R^(1′) and R^(2′) is independently selected from hydrogen,        hydroxyl, optionally substituted alkyl, optionally substituted        alkenyl, optionally substituted alkynyl, optionally substituted        alkoxy, optionally substituted carbocyclyl, optionally        substituted aryl, optionally substituted heterocyclyl,        optionally substituted heteroaryl, a ionizable side group, and a        functionalizable side group.

-   102. The method of 101, wherein the precursor polymer comprises    functionalizable repeat units of the formula (I′) wherein at least    one of the R^(1′) s and R^(2′)s is a functionalizable side group.

-   103. The method of 101 or 102, wherein the precursor polymer    comprises repeat units of the formula (I′) wherein each R¹′ and R²′    is independently selected from hydrogen, C₁₋₅ alkyl and a    functionalizable side group.

-   104. The method of any one of 101-103, wherein the precursor polymer    comprises repeat units of the formula (I′) wherein m′ is an integer    from 1 to 5.

-   105. The method of any one of 101-104, wherein the precursor polymer    comprises repeat units of the formula (I′) wherein one of the R¹′s    and R²′s is a functionalizable side group and all of the remaining    R¹′s and R²′s are not functionalizable side groups.

-   106. The method of any one of 101-105, wherein the precursor polymer    comprises repeat units of the formula (I′) wherein m′ is 5, one R¹′    is a functionalizable side group and all of the remaining R¹′s and    R²′s are hydrogen.

-   107. The method of any one of 101-106, wherein the precursor polymer    comprises repeat units of the formula (I′) wherein m′ is 5 and all    of the R¹′s and R²′s are hydrogen.

-   108. The method of any one of 101-107, wherein the precursor polymer    comprises repeat units of the formula (I′) wherein m′ is 1, R¹′ is    hydrogen and R²′ is hydrogen.

-   109. The method of any one of 101-108, wherein the precursor polymer    comprises repeat units of the formula (I′) wherein m′ is 1, R¹′ is    methyl and R²′ is hydrogen.

-   110. The method of any one of 101-109, wherein the precursor polymer    comprises repeat units of the formula (I′) wherein m′ is 2, the R¹′    and R²′ alpha to the carbonyl group in formula (I′) are each    hydrogen, the R¹′ beta to the carbonyl group is methyl and the R²′    beta to the carbonyl group is hydrogen.

-   111. The method of any one of 101-110, wherein the repeat units    comprise repeat units of the formula (I′) wherein m is 3 and all of    the R¹′s and R²′s are hydrogen.

-   112. The method of any one of 101-111, wherein the precursor polymer    comprises repeat units of the formula (I′) wherein m′ is 4, and all    of the R¹′s and R²′s are hydrogen.

-   113. The method of any one of 101-112, wherein the repeat units    comprise repeat units of the formula (I′) wherein m is 3, the R¹′    and R²′ alpha to the carbonyl group in formula (I′) are each    hydrogen, the R¹′ and R²′ beta to the carbonyl group are each    hydrogen, and the R^(1′) gamma to the carbonyl group is methyl and    the R²′ gamma to the carbonyl group is hydrogen.

-   114. The method of any one of 101-113, wherein the functionalizable    group is selected from a leaving group, an azido group or an alkynyl    group.

-   115. The method of any one of 101-114, wherein the precursor polymer    is a homopolymer of repeat units of formula (I′) wherein at least    one of the R¹′s and R²′s is a functionalizable side group.

-   116. The method of any one of 101-114, wherein the precursor polymer    is a copolymer comprising at least two different repeat units.

-   117. The method of 116, wherein each of said at least two different    repeat units is of formula (I′) and wherein at least one of said at    least two different repeat units has a formula (I′) in which at    least one of the R¹′s and R²′s is a functionalizable side group.

-   118. A complex as defined in any one of 1-55 for use as a    medicament.

-   119. A composition as defined in any one of 56-82 for use as a    medicament.

-   120. The composition of 67 or 68, wherein the vehicle polymer is a    biodegradable polymer.

-   121. A method comprising:    -   combining a functionalized polymer with a pharmaceutically        active agent, the functionalized polymer comprising ionizable        repeat units comprising at least one ionizable side group, to        form a complex in which a plurality of the at least one        ionizable side groups form a plurality of non-covalent bonds        with the pharmaceutically active agent.

-   122. The method of 121, comprising preparing the functionalized    polymer from a precursor polymer, the precursor polymer comprising    repeat units, the repeat units comprising functionalizable repeat    units comprising at least one functionalizable side group, wherein    the preparing comprises transforming, using click chemistry, said    functionalizable repeat units into ionizable repeat units comprising    at least one ionizable side group.

-   123. The complex of any one of 1-55, wherein the functionalized    polymer is not a polyamino acid.

What is claimed is:
 1. A complex comprising: a pharmaceutically activeagent, and a functionalized polymer, the functionalized polymercomprising repeat units, the repeat units comprising ionizable repeatunits comprising at least one ionizable side group, a plurality of theat least one ionizable side groups forming a plurality of non-covalentbonds with the pharmaceutically active agent, wherein at least 10% ofthe repeat units comprise at least one ionizable side group, and whereinthe ionizable repeat units comprise one or more ionizable side groupsthat comprise (1) an optionally substituted heteroarylene ring that is a1,2,3-triazole ring and (2) at least one member selected from ammonium,carboxylate, hydrazinium, guanidinium, sulfate, sulfonate, phosphonate,and phosphate.
 2. The complex of claim 1, wherein the percentage of therepeat units comprising at least one ionizable side group ranges from20% to 80%.
 3. The complex of claim 1, wherein the functionalizedpolymer is synthetic.
 4. The complex of claim 1, wherein thefunctionalized polymer is a polyester.
 5. The complex of claim 1,wherein the at least one ionizable side group is covalently bound to thepolymer through click chemistry.
 6. The complex of claim 1, wherein theplurality of non-covalent bonds comprise electrostatic interactions. 7.The complex of claim 1, wherein the pharmaceutically active agentcomprises at least one member selected from a peptide, protein, andsmall molecule, the small molecule having a molecular weight less than500 Daltons.
 8. The complex of claim 1, wherein the at least oneionizable side group comprises a negatively ionizable side group.
 9. Thecomplex of claim 1, wherein the functionalized polymer has a weightaverage molecular weight ranging from 1000 Daltons to 200,000 Daltons,as measured by gel permeation chromatography.
 10. The complex of claim1, wherein the functionalized polymer has a solubility in water of atleast 0.01 mg/ml and less than or equal to 10 mg/ml at 25° C. and pH7.4.
 11. The complex of claim 1, wherein the complex has a solubility ofless than 0.01 mg/mL in water at 25° C. at pH 7.4.
 12. The complex ofclaim 1, wherein the ratio of the amount of the pharmaceutically activeagent to the amount of the functionalized polymer in the complex is from1:1 to 1:10 by weight.
 13. The complex of claim 1, wherein thefunctionalized polymer is not a polyamino acid.
 14. The complex of claim1, wherein the functionalized polymer is linear.
 15. The complex ofclaim 1, wherein the functionalized polymer has a weight averagemolecular weight greater than 15,000 Daltons, as measured by gelpermeation chromatography.
 16. The complex of claim 1, wherein at least40% of the repeat units comprise at least one ionizable side group.